Gahan 2013 Acidos Nucleicos Efectos Inherencia

7/27/2019 Gahan 2013 Acidos Nucleicos Efectos Inherencia

1/18

REVIEW ARTICLE

Circulating nucleic acids: possible inherited effects

PETER GAHAN*

Anatomy & Human Sciences, Kings College London, London Bridge, London SE1 1UL, UK

Received 17 June 2013; revised 8 July 2013; accepted for publication 8 July 2013

The presence of circulating nucleic acids in man, animals and plants is well documented. It is clear that suchnucleic acids can not only circulate freely within an organism, but can also enter cells when their biology may bechanged either epigenetically or genetically. Evidence is presented concerning a possible influence of these nucleic

acid fragments on the genetics of the F1 generation of man, animals and plants. The data presented also offer amechanism by which the incorporation of horizontally transferred genes between organisms may be achieved. Therole that circulating nucleic acids might play in modifying the F1 generation and possibly the evolutionary processis considered. 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, , .

ADDITIONAL KEYWORDS: animals circulating nucleic acids epiphytes eutheria horizontal genetransfer man ova parasites sperm vascular plants.

INTRODUCTION

The nucleic acids present in the circulatory system of

eukaryote organisms include genome sequences

derived through controlled release and/or clearance of

such fragments from cells. These fragments are able to

move around the organism and to enter other cells

where they may change the biology of the recipient

cells (Zamecnik et al., 1994; Gahan & Stroun, 2010a).

This may be an important aspect for the field of

epigenetic variations (Gahan & Stroun, 2010a; Peters

& Pretorius, 2011). However, there is a need to ques-

tion the assumption that biological variations are

limited to epigenetic events only. This is a result of

some nucleic acid fragments passing into the germ-line

cells and hence into the offspring, sometimes leading

to non-Mendelian inheritance (Yagishita, 1961a, b;

Stroun, 1962; Stroun, Mathon & Stroun, 1963a). Such

inheritance is clearly shown by grafting experiments

in plants. Although heterografts can lead to epigenetic

changes in the offspring, homografts can lead to

genetic inheritance (Yagishita, 1961a, b; Stroun, 1962;

Stroun et al., 1963a). This was assumed to result from

the movement of DNA from the host to the graft.Similarly, Stroun et al. (1958, 1963b) demonstrated

that birds of the white leghorn variety, which were

repeatedly injected with blood from the grey guinea

fowl, produced progeny with some grey or black-

flecked feathers in the second and later generations.

Nucleic acids readily pass by horizontal transfer

between prokaryotes (Davis et al., 1973; Dunning

Hotopp et al., 2007; Nosenko & Bhattacharya, 2007),

between prokaryotes and eukaryotes (Heinemann &

Sprague, 1989; Doolittle et al., 1990; Scholl et al.,

2003; Morrison et al., 2007; Craig et al., 2008;

Gladishev, Meselson & Arkhipova, 2008), between

eukaryotes and prokaryotes (Nielsen et al., 1998;Pontoroli et al., 2009; Demanche et al., 2011),

and between eukaryotes (Montes et al., 2003;

Bergthorsson et al., 2004; Davis & Wurdack, 2004;

Davis, Anderson & Wurdack, 2005; Barkman et al.,

2007; Park, Manen & Schneeweiss, 2007; Richardson

& Palmer, 2007; Gladishev et al., 2008; Keeling &

Palmer, 2008; Nedelcu et al., 2008; Rumpho et al.,

2008; Whitaker, McConkey & Westhead, 2009; Gilbert

et al., 2010; Mower et al., 2010; Yoshida et al.,

2010).*E-mail: [email protected]

bs_bs_banner

Biological Journal of the Linnean Society, 2013, , .

2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, , 1


2/18

Circulating nucleic acids (CNAs), both DNA and

RNAs, can exert both epigenetic and genetic changes,

thus offering an explanation for the way in which

horizontally-transferred genes may influence the off-

spring of the recipient eukaryote.

Consequently, there is a need to revisit the concept

of the horizontal gene transfer (HGT) aspect of the

CNAs leading to both genetic and epigenetic effects.

The present review considers the involvement of

total CNAs in eukaryotes in the induction of both

epigenetic and genetic changes in eukaryote organ-isms. Furthermore, it examines a possible role for

these nucleic acid fragments in the genetics of the F1generation and, hence, possibly in the evolutionary

process.

ORIGINS OF CIRCULATING NUCLEIC ACIDS

The release of nucleic acids into the prokaryote envi-

ronment appears to be limited to prokaryote cell

breakdown and nucleic acid release, whereas various

specific mechanisms exist for DNA transfer between

individual prokaryotes (Davis et al., 1973; Nosenko &

Bhattacharya, 2007). However, the origin of the CNAsin eukaryotes is manifold. Thus, there are at least

eleven possible endogenous sources of both RNA and

DNA in the man/animal (blood) circulatory system

(Table 1) and at least nine endogenous sources

of plant CNAs (Table 2). In the case of plants,

although the soil contains a variety of DNAs, they are

unlikely to enter the circulatory system because they

will be degraded upon entry to the root system

(Gahan et al., 1968, 1974; Paungfoo-Lonhienne et al.,

2010).

CAN CNAS BE INVOLVED IN MODIFYINGTHE F1 GENERATION AND,

SUBSEQUENTLY, THEEVOLUTIONARY PROCESS?

For CNAs to operate in a way that will influence

evolution, it is necessary that such nucleic acid

fragments/molecules can freely access cells into which

they enter and modify the biology of the recipient cell.

For there to be a modification of the F1 generation and

an evolutionary implication, the DNA must be able to:

(1) access the gonads; (2) directly enter either sperm

and eggs or their precursor cells; and (3) integrateinto (a) chromosome(s) so affecting the offspring.

The experimental evidence for the possible involve-

ment of DNA in genetic changes can be seen with

experiments on both vascular plants and animals

with circulatory systems. However, there are major

differences between the plant and animal reproduc-

tive systems in that the majority of animals have

a distinct germ cell line separate from the soma

(Weissman, 1893; Maynard Smith, 1969, 1989),

whereas vascular plants develop reproductive

systems from vegetative cells on each reproduc-

tive occasion (Esau, 1953; Fahn, 1982). Equally,

pteridophyta produce sexual reproductive organs andsporogonia from vegetative cells, as do bryophytes

with their more primitive water conducting tissues,

whereas the gymnosperma also derive their sexual

organs from vegetative cells. The change from veg-

etative to reproductive cells, i.e. no germ cell line, in

plants is, perhaps, one of the strongest arguments

against Weissmans general theory (Maynard Smith,

1969, 1989).

Hence, the mechanisms of CNA entry into reproduc-

tive systems will be considered separately primarily

Table 1. Possible origins of circulating nucleic acids in

man/animals

DNA and RNAs*

1. Blood cell breakdown

2. Bacteria breakdown

3. Viruses

4. Cell and tissue necrosis

5. Cell apoptosis

6. Cellular release of exosomes

7. Cellular release of siRNA

8. Cellular release of virtosomes

9. Parasite nucleic acids

10. Mitochondria

DNA

1. Cellular release of transposons and retrotransposons

2. Leukocyte surface DNA

*mRNA, small-interfering (si)RNA, microRNA, RNAi,

double-stranded RNA, nanoRNA, coding RNA, long

noncoding RNA.

Table 2. Possible origins of circulating nucleic acids in

vascular plants

DNA and RNAs*

1. Bacteria breakdown and viruses

2. Differentiation of tracheids

3. Differentiation of vessels

4. Differentiation of sieve elements

5. Cellular release of siRNA

6. Cellular release of virtosomes

7. Mitochondria

8. Parasites

9. Epiphytes

DNA

10. Cellular release of transposons and retrotransposons

*mRNA, small-interfering (si)RNA, microRNA, RNAi,

double-stranded RNA, nanoRNA, coding RNA, long

noncoding RNA.

2 P. GAHAN

2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, ,


3/18

for vascular plant systems through the angiosperma

and man/animals through the eutheria for which most

information is available. This will lead to a better

understanding of the possible influence of CNAs on the

F1 generations and, subsequently, evolution.

POSSIBLE EPIGENETIC CHANGES ON CNAENTRY INTO EUTHERIAN SOMATIC CELLS

Extracellular nucleic acid fragments from many

sources (Table 1) are capable of entering other cells in

the same organism and changing the biology of the

recipient cells.

Examples exist demonstrating the relative ease of

cell uptake of nucleic acids circulating throughout the

organism and the likely ability of such fragments to

exert their effect through an epigenetic mechanism as

suggested by Peters & Pretorius (2011). However, a

genetic involvement cannot be ruled out in some casesbecause the experiments to test such situations have

not been performed.

The effect of the uptake of DNA and RNA on cell

division can be seen in the studies of two mouse

tumour cell lines, J774 and P497, and nonstimulated

lymphocytes (Adams, Diaz & Gahan, 1997). The

newly-synthesized DNARNAlipoprotein complex,

the virtosomes (Gahan & Stroun, 2010b), released

from the tumour cell lines stimulated nuclear DNA

synthesis in approximately 60% of nonstimulated

lymphocytes, whereas those from the nonstimulated

lymphocytes and nondividing hepatocytes inhibited

DNA synthesis in approximately 60% of tumour celllines. Similar reciprocal events occur between stimu-

lated and nonstimulated lymphocytes (Viola-Magni

et al., 2011), whereas nondividing hepatocyte virto-

somes blocked the cell division of SW 480 tumour

cells to differing degrees dependent upon the concen-

tration of virtosomes employed (M. Garcia-Arranz,

D. Garcia-Olmo, L. Vega, M. Stroun, P. B. Gahan,

unpubl. data). Furthermore, glioblastoma-derived

vesicles containing mRNA have been shown to stimu-

late the proliferation of a human glioblastoma cell

line (Skog et al., 2008).

DNA released from X-irradiated Chinese hamster

ovarian cells initiated similar cytological changes onentering non-irradiated cells (i.e. the transposition of

chromosomal peri-centrimeric loci of homologous

chromosomes from the peri-membrane sites to

approach each other). In addition, nucleolar forming

chromosome regions were activated (Ermakov et al.,

2008). Radiation-induced bystander effects (RIBEs)

can be defined as the responses of non-irradiated cells

to the molecular events occurring in their irradiated

neighbours having been observed both in vivo and in

cultured human/animal cells (Klokov et al., 2004;

Hamada et al., 2007; Hei et al., 2008; Matsumoto

et al., 2011). When human tissues were irradiated

locally, RIBEs were apparent in both adjacent areas

and in cells distant from the beam. In some cases, an

increased number of double-strand breaks have been

found in the bystander cells together with DNA

damage response, genomic instability, and decreasedcell viability (Lorimore & Wright, 2003; Trainor et al.,

2012).

Two different DNA fragments isolated from the

blood of myocardial infarct patients have been found

to alter the contraction frequency of neonatal rat

ventricular myocytes in vitro. Thus, the AT-rich frag-

ments of human satellite three tandem repeat (1 q12

space region) increased the contraction rate, whereas

the GC-rich fragments of rDNA decreased the con-

traction rate (Bulicheva et al., 2008). During heart

failure, mitochondrial DNA is released into heart

tissue through cell breakdown and can kickstart the

bodys natural response to infection, thus contributingto the process of heart failure. During heart failure,

immune cells invade the heart leading to inflamma-

tion and less efficient heart muscle, reducing its

ability to pump blood around the body. Inflammation

is usually only activated (e.g. on infection by bacteria

or viruses). Because mitochondria are of bacterial

origin, their DNA, resembling that of bacteria, initi-

ates inflammation by triggering Toll-like receptor 9 in

the immune cells (Oka et al., 2012).

Immune responses have been recorded in addition

to the classical Toll system initiation of the process

(Hemmi et al., 2000; Dalpke et al., 2006). An allogenic

TB lymphocyte co-operation in the presence of thesupernatant from the culture medium of T cells, pre-

viously exposed to inactivated herpes simplex virus,

synthesized an anti-herpetic antibody carrying some

allotypic markers of the T cell donor. DNA purified

from the supernatant of the T cell culture medium

also had the same effect on B lymphocytes (Anker

et al., 1980).

In other experiments (Anker et al., 1984), nude

mice were injected with DNA extracted from the DNA

complex released by human T lymphocytes previously

exposed to inactivated herpes or polioviruses. The

serum from these mice, tested for its neutralizing

activity, showed that mice synthesized anti-herpeticor anti-polio antibodies, respectively. The antibodies

carried human allotypes, as shown through their neu-

tralization by human anti-allotype sera.

POSSIBLE GENETIC CHANGES ON CNAENTRY INTO EUTHERIAN SOMATIC CELLS

In the examples of either DNA or RNA uptake by cells

given above, the changes observed can most likely be

CIRCULATING NUCLEIC ACIDS 3



4/18

explained by epigenetic events. Nevertheless, some

experiments imply that there is an integration of the

DNA fragment into the genome and hence a genetic

effect can be established. Indeed, this is one possible

mechanism that has been suggested for the induc-

tion of metastases (Garca-Olmo et al., 1999; 2000;

Garcia-Olmo, Ruiz-Piueras & Garcia-Olmo 2004;Garcia-Olmo et al., 2010).

Studies of the release of mutated Kras fragments of

SW 480 cells showed them to be readily taken up by

NIH 3T3 cells, leading to their transformation (Anker

et al., 1994). Furthermore, NIH 3T3 cells were also

transformed by mutated Kras sequences on culturing

with the plasma from patients with Kras mutated

colorectal tumours. These oncogenetically trans-

formed 3T3 cells initiated carcinomas in NOD-SCID

mice, with such tumours being found to contain the

human mutated Kras sequence (Garcia-Olmo et al.,

2010) that was also found free in the mouse blood. A

similar outcome has been reported by Trejo-Becerrilet al. (2012) when employing the same experimental

system as Garcia-Olmo et al. (2010). Additionally,

Garcia-Olmo et al. (2012) also demonstrated that

tumour DNA was found in the plasma during a 2-year

follow-up period after surgical removal of the

colorectal tumour from patients. This DNA also led to

similar results with the same experimental system

involving NIH 3T3 cells and NOD-SCID mice

(Garcia-Olmo et al., 2012).

Based upon data indicating that chromatin frag-

ments possessing recombinagenic free ends were

present in the plasma and serum, they could be

exploited in gene replacement therapy. Small frag-ments were prepared of human chromatin from non-

mutant cells and added to the culture medium of

human breast cancer cells having a 47-bp deletion in

the CASP 3 gene. Caspase 3 activity was restored in

30% of the treated cells (Yakubov et al., 2007).

The phagocytotic uptake of apoptotic bodies led to

the transfer of genomic DNA to the nuclei of the

recipient cells, with such DNA being stable over time

(Holmgren et al., 1999). Further involvement of DNA

uptake leading to tumorigenesis can be seen through

experiments exploiting the phagocytosis of apoptotic

bodies derived from H-rasV12- and human C-myc-

transfected rat fibroblasts by p53-deficient fibroblastsin vitro. The transferred DNA was propagated in the

recipient cells that also have been found to contain

either rat chromosome(s) or rat and mouse fusion

chromosomes in their nuclei (Bergsmedh et al., 2001).

MECHANISMS OF RNA AND DNA UPTAKEBY EUTHERIAN CELLS

On examining the mechanisms of eutherian cell CNA

uptake, two aspects need consideration: (i) entry into

the cytoplasm without CNA destruction and (ii) move-

ment from the cytoplasm into the nucleus where it

can act either epigenetically or be incorporated into a

chromosome.

ENTRY INTO THE CYTOPLASM

Very few studies have been made on the mechanism

of RNA uptake. Given that most RNA released from

cells tends to be via exosomes, a likely explanation for

the uptake of exosomal RNA is by endocytosis. This is

supported by studies in plants on small-interfering

(si)RNA in which the endolysosomal system is consid-

ered to be involved (Gibbings & Voinnet, 2010). Nev-

ertheless, in Drosophila cells, dsRNA uptake from the

environment requires receptor-mediated endocytosis

(Saleh et al., 2006). Lee et al. (2009) have linked gene

silencing by microRNAs and siRNAs to endosomal

trafficking.

The molecular mechanisms of DNA uptake in mam-malian cells are poorly understood (Wittrup et al.,

2007). However, available studies indicate that the

uptake of virtosomal DNA and other DNAs is more

complicated than that proposed above for RNA.

Although various sources of DNA such as bacterial

and mitochondrial DNA, have been shown to enter

cells by the Toll receptor system (Hemmi et al., 2000,

2002; Dalpke et al., 2006; Oka et al., 2012) there is

every reason to assume that DNAs can enter cells by

using alternative mechanisms. The question arises as

to how these mechanisms use processes that avoid the

lysosomal digestive system. This is not clear with

respect to the phagocytic uptake of apoptoticbodies (Bergsmedh et al., 2001). The relatively few

studies that have been performed on mouse skeletal

muscle (Wolff et al., 1990), human keratinocytes

(Basner-Tschakarjan et al., 2004), J774 cells

(Trombone et al., 2007), and murine GEnC cells

(Haegle et al., 2009) indicated a variety of methods

involving T tubules (Wolff et al., 1990), caveoli (Wolff

et al., 1990; Zamecnik et al., 1994) and endosomes

(Yakubov et al., 1989; Trombone et al., 2007; Haegle

et al., 2009). In particular, the uptake of naked

plasmid DNA via endosomes has been shown to result

in the blocking of endosomal acidification, thus avoid-

ing activation of the hydrolases present and henceDNA digestion (Trombone et al., 2007). Such DNA

remains in the endosomes to be transported to the

nuclear membrane. The authors suggest that the

DNA is not released from the vesicles but may be

transferred directly into the nucleus. Endocytosis

and pinocytosis have also been implicated by Wagner

et al. (1990), Luo & Saltzman (2000) and Basner-

Tschakarjan et al. (2004). This may be a mechanism

by which the virtosomes enter the cell, the virtosome-

containing endosomes avoiding the lysosomal system.

4 P. GAHAN



5/18

Caveoli have been suggested as an alternative entry

mechanism for nucleic acids to avoid lysosomal diges-

tion via potocytosis. There are at least four different

caveoli membrane traffic patterns with at least four

locations for caveoli ligands (Anderson et al., 1992;

Mineo & Anderson, 2001). However, there is some

evidence that the caveoli may eventually link withthe lysosomal system (Kiss & Botos, 2009; Kiss,

2012).

Alternative mechanisms considered in a number of

studies have demonstrated that histones H1 (Fritz

et al., 1996; Budker et al., 1997; Zaitsev et al., 1997;

Bottger et al., 1998; Haberland et al., 2000; Balicki

et al., 2002), H2A (Singh & Rigby, 1996; Balicki &

Beutler, 1997; Balicki et al., 2000), and H3 and H4

(Demirhan et al., 1998; Hariton-Gazal et al., 2003) are

effective mediators of transfection. The postulated

mechanisms by which histone H1 increases gene

transfection are through DNA condensation, DNase

protection, and the mediation of nuclear import. Inthese studies, histone H1 was considered to be the

subclass of histones that mediates efficient gene

transfer in the presence of chloroquine. Balicki et al.

(2002) have further determined that DNA-delivery

activity can be mediated by two mechanisms: electro-

statically driven DNA binding and condensation by

histone and nuclear import of these histone H2ADNA

polyplexes via nuclear localization signals in the

protein. More recently, Peters & Pretorius (2011) have

also suggested that, because histones can increase the

permeability of membranes by ionic interaction, this

mechanism could aid complexes, such nucleosomes

released by apoptosis, to enter recipient cells. It hasbeen suggested that virtosomes could enter cells by

one of the mechanisms described above (Gahan &

Stroun, 2010b). Virtosomes are comprised of DNA,

RNA and glycolipoprotein and do not appear to either

pick up or lose membrane material on either leaving

or entering cells. Therefore, it is possible that a

mechanism similar to that exploited by histones could

lead to the direct uptake of virtosomes through an

ionic interaction between a part of the glycoli-

poprotein present and the cell membrane.

This proposition is further supported by the work of

Wittrup et al. (2007) who showed that naked plasmid

DNA uptake does occur via proteoglycan-dependentmacropinocytosis. Such data challenge the concept

involving a specific DNA-internalizing receptor.

NUCLEAR UPTAKE OF NUCLEIC ACIDS

Having entered the cytoplasm, the nucleic acid must

enter the nucleus if it is to have either a genetic or an

epigenetic effect. The nuclear envelope contains

nuclear pores with a passive transport limit of 70 kDa

molecular mass or 10 nm diameter (Melchior &

Gerace, 1995). Hence, the nuclear membrane presents

a considerable barrier to the entry of nucleic acids.

Nevertheless, DNA can be seen to enter the nucleus of

chick embryo fibroblasts (Adams & Macintosh, 1985;

Zamecnik et al., 1994), HeLa cells (Zamecnik et al.,

1994), L29 mouse fibroblasts and Krebs 2 ascites

carcinoma cells (Yakubov et al., 1989). Furthermore,the nuclear sites occupied by the 3H-oligonucleotides

used by Zamecnik et al. (1994) essentially differed

from those occupied by the incorporated 3H-

thymidine as observed by transmission electron

microscopy autoradiography. The mechanism by

which DNA enters the nucleus is not clear. It is

known that, for mediated active transport through

the nuclear pore complex, nuclear proteins require a

nuclear localization signal that contains basic amino

acids and can be recognized by cytosolic factors (Jans

& Hubner, 1996). For this to occur, the nuclear pore

can expand to approximately 30 nm (Dworetzky,

Lanford & Feldherr, 1988). For example, this can beshown to function experimentally by the coupling of

100 nuclear localization signal peptides/kilobase pair

of DNA for the nuclear delivery of the DNA

(Sebestyen et al., 1998).

RNA movement from cytoplasm to nucleus appears

to involve specific proteins. Thus, in the case of

siRNAs, there is the necessity for them to be linked to

an argonaut protein for transfer to the nucleus (e.g.

NRDE-3 in Caenorhabditis elegans) (Guang et al.,

2008).

MITOCHONDRIAL RELEASE AND UPTAKEOF NUCLEIC ACIDS

Little is known about the release of DNA from animal

mitochondria, other than through cell death, and its

uptake into mitochondria has not been as well studied

for animals as it has for plants. A mitochondrial

permeability transition pore complex existing in asso-

ciation with the inner mitochondrial membrane has

been well studied (Bernardi, 2013). Although it

permits the passage of molecules of up to 1500 Da,

currently there is no evidence for a similar passage of

DNA to that seen with plant mitochondria (see

below).

NUCLEIC ACID UPTAKE INTO EUTHERIANOVA AND SPERM

The information is less substantiated for man/

animals than for plants but, nevertheless, it leads to

the strong possibility of the influence of CNAs on the

progeny. The earliest experiments indicating the pos-

sibility that circulating DNA might be incorporated

into ova and/or sperm of the recipient organism came




6/18

from the initial studies by Benoit et al. during the

1950s and 1960s on the pekin duck. DNA isolated

from the nuclei of genital organs of khaki ducks

(greenishblack beaks) was injected into nine females

and three males of the pekin (yellow beak) variety.

The progeny showed a marked reduction in weight,

modified feathers and carriage. Three generationsderived by either inter se matings or from back-

crosses to pekins yielded several hundred female

descendents. Of these, 18 had bluish markings on the

beaks from the age of 4 months, being more pro-

nounced in the breeding season. Treated male parents

and several of their male progeny developed

draughts board-like markings on the web of the foot

(Benoit et al., 1960, 1966). These results have often

been criticized because there was no attempt to

repeat the experiments and, given the methods avail-

able at the time, there was the possibility that the

DNA preparation was impure. However, subsequent

work using purified and labelled DNA has tended tosupport the possibility of the transfer of DNA to the

gonads. Moreover, the introduction of DNA fragments

into sperm forms the basis of the method for achiev-

ing genetic modifications in domestic animals

(Brackett et al., 1971; Niu & Liang, 2008).

OVA

The idea that circulating DNA might have ready

access to the female gonads is supported by the early

experiments of Ledoux & Charles (1970) when, within

1 h of injecting mice intravenously with 3H-DNA fromEscherichia coli, the 3H-DNA was incorporated in

ovarian tissues as confirmed by both CsCl centrifu-

gation and autoradiography; in the latter case, the

oocyte nuclei were labelled. Little labelling appeared

to be retained by the uterine and vaginal tissues.

However, intravenous injection of 3H-DNA into preg-

nant mice led to labelling of the embryos especially in

the early stages of development.

Liu (2006) identified 50 reports on blood transfu-

sion experiments (parabiosis), performed between

1950 and 1979, and involving a variety of birds, pigs,

and rabbits of which 45 yielded positive results and

only five obtained negative results. A successfulexample of parabiosis concerns an experiment to

investigate heritable changes in a 50-day old Angora

female parabiosed for 36 days with a Flanders female

of similar age (Boriachok-Nizhnik, 1951). One month

after separation, the Angora female was mated to an

Angora male, yielding seven offspring of which only

one had Angora long hair, with the other six having

Flanders-like short hair. The young rabbits had a

mean weight of 3020 g at 5 months compared to

2330 g for normal Angora rabbits under similar con-

ditions. This implied that DNA fragments circulating

in plasma and serum can influence ova in the experi-

mental animals employed.

Subsequently, there has been little work on the

introduction of DNA into ovaries/ova (Guerra,

Carballada & Esponda, 2005; Niu & Liang, 2008).

Although chronic hepatitis B virus has been shown toinfect ova at different stages of development (Ye et al.,

2005), the transfection of ovarian cells is considered

to be difficult. Yang et al. (2007) did manage to

transfect ovarian cells with enhanced green fluores-

cent protein (EGFP)-expressing plasmid (pIRES-

EGFP) by direct injection into the ovaries of fertile

mice. Following natural fertilization, healthy trans-

genic mice were obtained, with the introduced EGFP

gene being inherited by 64.9% of the F1 offspring and

with stable transmission to 66.94% of the F2 progeny.

The foreign genes were both integrated into the

genome (frequency of 85.71% by FISH analysis) and

translated into the functional protein on transferringto the subsequent generation. Hamster oocytes

incubated in vitro with human papilloma viral DNA

passively took up exogenous DNA fragments possibly

by either endocytosis or pinocytosis (Wagner et al.,

1990; Luo & Saltzman, 2000). Transfection studies

directly using the bloodstream to carry DNA to the

oocytes in mouse ovaries have proved successful

(Tsukamoto et al., 1995) as demonstrated by the

modified offspring.

Given that follicles housing the primary oocytes are

supplied by peripheral blood capillaries, CNAs need

to traverse the various follicular layers between the

blood supply and the primary oocyte. Nevertheless,there are mechanisms by which CNAs are found to be

present in the primary oocytes (Wagner et al., 1990;

Tsukamoto et al., 1995; Luo & Saltzman, 2000; Ye

et al., 2005; Yang et al., 2007). Although there is a

very low probability that a transformed primary

oocyte will be fertilized by a transformed sperm, there

is a greater probability that a transformed primary

oocyte could be fertilized by a nontransformed sperm,

or vice versa. This is especially so for invertebrates or

vertebrates that lay their eggs in large numbers as

opposed to when the eggs are laid singly. Hence, for

eutherians, with some having a long reproductive life,

primary oocytes may be released in either smallnumbers or singly, thus limiting the chances of the

fertilization of a transformed primary oocyte.

In addition, the primary oocyte chromosomes will

be arrested at diplotene of the first meiotic division,

the dictyate phase, and will remain so until their

release from the follicle and fertilization by the sperm

(Snell, 1972). Hence, there appears to be only a short

window of time in which the integration of circulating

DNA fragments into the oocyte nucleus can occur (i.e.

at around the time of fertilization when the meiotic

6 P. GAHAN



7/18

block is released). Consequently, it is more likely that

any transformation of the primary oocytes by circu-

lating DNA fragments will need to occur during

embryogenesis when they must enter the chromo-

somes of cells involved in the production of oogonia

that (through successive mitoses) form the primary

oocytes.For example, in the case of man, there is only a

small chance of a transformed primary oocyte being

selected in the human female. Not all of the approxi-

mately 70 000 primary oocytes present in the

3-month-old embryo are likely to be transformed by

CNAs prior to being reduced through atresia to

approximately 40 000 at puberty. In addition to their

continuing to atrese during the reproductive lifetime

of the female, only some 400 primary oocytes will be

exploited during the female reproductive period, not

all of which may have been transformed. Of these,

only a very small number will be fertilized.

Although preliminary studies have permitted theisolation of putative oocyte stem cells from reproduc-

tive human and mouse ovaries (Johnson et al., 2004;

Zhou et al., 2009; White et al., 2012), it is not clear as

to how much of an active role they play in vivo in

adult egg production.

SPERM

Sperm present a different situation to ovaries and

ova. Recent research has involved sperm as gene

carriers for the production of transgenic animals

(Brackett et al., 1971; Chang et al., 1999; Smith &

Spadafora, 2005; Niu & Liang, 2008). In this situa-tion, direct DNA uptake by sperm appears to be

limited by the presence of DNAase and DNA-binding

proteins in the seminal fluid, possibly released by

the prostate (Carballada & Esponada, 2001; Lanes,

Sampaio & Marins, 2009). However, if the sperm are

washed well, they are capable of taking up DNA

directly even into the nucleus and the mitochondria

(Brackett et al., 1971; Spadofora, 1998; Guerra et al.,

2005; Vasicek et al., 2007; Niu & Liang, 2008). An

alternative approach to introduce DNA into sperm

has been by direct injection of DNA encapsulated in

liposomes into the testes via the scrotum (Chang

et al., 1999). This exogenous DNA appears to berapidly transported to the epididymal ducts via the

rete testis and efferent ducts to be incorporated by

epithelial cells of the epididymus and epididymal

spermatozoa (Sato, Ishikawa & Kimura, 2002).

Sperm cells incubated with human poliovirus RNA

take up the exogenous RNA molecules and internalize

them in the nuclei. The poliovirus RNA is reverse-

transcribed in coding DNA (cDNA) fragments that

can be visualized on sperm nuclear scaffolds by

immunogold electron microscopy. The poliovirus

RNA-challenged spermatozoa are able, subsequently,

to transfer the retro-transcribed cDNA molecules into

eggs during in vitro fertilization (Giordano et al.,

2000).

As with oocytes, sperm uptake of CNA fragments

would preferably occur during spermatogenesis when

cells will be more amenable to uptake. In the vastmajority of cases where there is only one or very few

reproductive periods in the year, all sperm will be

released at once, thus giving rise to the possibility of

any transformed sperm being able to fertilize a(n)

(un)transformed oocyte. However, in man, when there

is continuous sperm production, there will be a con-

tinual turnover of sperm. The experimentally deter-

mined time from sperm production to ejaculation in

healthy men is 4276 days (Missel et al., 2009).

Hence, given the high turnover rate of sperm and the

fact that, in man, up to 100 millions of sperm are

released at each ejaculation, there is a very low

probability of a CNA transformed sperm fertilizing aCNA (un)transformed oocyte.

CNA UPTAKE IN EUTHERIA ANDTHEIR PROGENY

Both sperm and ova appear to be able to take up

nucleic acid fragments and, in the case of sperm,

transfer them to the ova. However, the nucleic acid

fragments already present in the bloodstream,

although having access to sperm and ova and hence

incorporation into chromosomes, have not so far been

demonstrated to have a major impact in terms ofmodifying the subsequent generations of such indi-

viduals. One reason is that eukaryoteeukaryote

transfer of nuclear genes is underestimated for various

reasons, including the fact that many of the acquired

genes are existing homologues rather than genes intro-

ducing new functions (Keeling & Palmer, 2008).

One way in which genes capable of modifying

organisms could enter the bloodstream would be from

an external source through either a bacterial/viral

infection or parasitic infections by, for example,

protozoa, platyhelminths, nematodes, annelids, and

insects. Currently, there is no evidence in support of

this idea (Brindley et al., 2009) in part because nostudies appear to have examined such a possibility.

Moreover, Beck et al. (2009) indicated that, in healthy

human individuals, no more than approximately 3%

of the circulating DNA is of nonhost origin and it is

not clear by how much such additional foreign DNA

could be present.

Nevertheless, both oocytes and sperm can be sup-

plied with CNAs and so take up nucleic acid frag-

ments. From the current evidence, sperm appear to be

the more likely carriers of such fragments that permit




8/18

the genetic changes necessary to affect the F1 genera-

tion. Hence, although there are mechanisms facilitat-

ing the nucleic acids present in the circulatory system

to make a strong impact on the F1 generation, to date,

there is little evidence that this is actually the case.

This could explain why there are so few examples of

horizontally-transferred genes being incorporatedinto gametes of eutheria and other higher animal

systems rather than into their somatic cells. This also

has relevance as to why, when genetically modified

DNA can pass from the gut to the circulatory system,

there has been no clear demonstration of the effects of

such DNA on the offspring of individuals eating

genetically modified food.

NUCLEIC ACID UPTAKE BY VASCULARPLANTS AND THEIR PROGENY

There appears to be a stronger case for the modifica-

tion of the progeny in plants on the uptake of CNAs

(Table 2) because plants develop floral reproductive

systems from vegetative sources on each reproductive

occasion rather than having a continuous germ cell

line as in many animals. Hence, there is ample oppor-

tunity for any CNA to integrate into the genome of

the vegetative cells that will ultimately be involved in

the formation of gametes. Modification of the genome

by siRNA generated internally has been considered

as a possible mechanism for changing the genome

expression in the F1 generation through incorporation

into primary meristem cells that are the precursors

of the reproductive organs (Melnyk, Molnar &

Baulcombe, 2011). However, this is self-generated

nucleic acid that will have an epigenetic effect rather

than a genetic one.

GRAFT HYBRIDS: EVIDENCE OFSPONTANEOUS TRANSFER OF NUCLEIC

ACIDS BETWEEN STOCK AND SCION

Initial studies on the grafting of plants by Darwin

(1868) and Mitchurin (1949) demonstrated the

passage of genetically retained characters from the

stock (mentor) to the scion (pupil) and, subsequently,

such results were repeated in several studies (Liu,2006). These include those of Burbank (191415) in

the USA with plum and Glouchtchenko (1948) in the

USSR with tomato. There were many earlier reports

of such experiments from China, although these were

not so well documented and, subsequently, from the

USSR (Liu, 2006).

Detailed grafting experiments were repeated in

Switzerland after discussion with Glouchtchenko

(Stroun, 1962; Stroun et al., 1963a) using Solanum

nigrum and two different varieties of Solanum

melanogena. It is important to note that these graft-

ing experiments were precursors to the development

of the idea for circulation of CNAs in higher organ-

isms, leading to their current use in the non-invasive,

early detection of foetal abnormalities and certain

diseases, as well as for monitoring treatment and

predicting clinical outcomes (Gahan, 2012, 2013).Their experiments consisted of grafting plants of

different varieties (S. melongena and S. nigrum) onto

one another. Either the stock or the scion was

deprived of all growing leaves and thus was subjected

to the influence of the metabolism of the leaf-bearing

section. The descendents of the underprivileged

partner of the scion (the pupil) sometimes demon-

strated genetically modified characteristics that were

often similar to those of the stock (the mentor).

However, the ways in which these modifications

appeared and were transmitted to the offspring were

often very different from those observed by sexual

crossing of the two varieties: (1) some modifications inthe pupil plant were similar to the characteristics of

the mentor plant; (2) characteristics not observed in

the mentor plant appeared in the pupil plant; (3) not

all of the modified pupil plants acquired the same

characteristics of the mentor plant, with some dem-

onstrating only one characteristic, others demonstrat-

ing several characteristics, or others demonstrating

all of the characteristics of the mentor; (4) during

segregation, some recessive parents produced off-

spring with dominant features; (5) segregation

occurred as early as the F1 generation of the modified

plants contrary to the expectation in sexual crossing

where segregation appears in the F2 generation; and(6) linked characteristics in the mentor plant some-

times appeared individually in the pupil plant and its

offspring. Similar results were obtained through

grafting between S. melanogena and S. nigrum.

The involvement of some kind of plasmic episomes

could be ruled out because (1) an accidental sex-

ual crossing can be absolutely excluded in view

of the aberrant segregation observed and that

S. melanogena and S. nigrum are not sexually com-

patible; (2) the number of alterations argues strongly

against the possibility of either mutations or specific

circulating viruses; and (3) the modifications appear-

ing in the pupil plants are carried by both the maleand the female gametes, as demonstrated by the

backcrossing of the modified plants. Alternative pos-

sibilities were offered to relate these unorthodox

results to classical genetics, namely:

1. Movement of genetic information from the mentor

plant to the pupil plant appeared to be the best

explanation for these phenomena and the possible

induction of apparent epigenetic modifications in

the fruit borne by the pupil.

8 P. GAHAN



9/18

2. In addition, given the appearance of the modifica-

tions in the F1 generation, the genetic transforma-

tion could be a result of the migration of DNA

released by the mentor cells to the somatic and

reproductive cells of the pupil plant.

At the same time, Yagishita (1961a, b) performedsimilar experiments using Capsicum baccatum and

Capsicum annuum and obtained similar results,

including the non-Mendelian segregation of the

new features in the progeny of the grafts. In addi-

tion, Hirata (1979, 1980, 1986) also worked on

S. melongena, obtaining similar results to those of

Stroun (1962) and Stroun et al. (1963a). They arrived

at similar conclusions in that there was a movement

of genetic material between the stock and the scion,

although Hirata et al. (2001) suggested that this was

through the action of retro-transposons. A similar

explanation was offered for the results obtained

through graft transformation in C. annuum (Hirataet al., 2003). Using male sterile petunia for stocks and

normal fertile petunia for scions, graft-induced

genetic variation was also demonstrated by Frankel

(1954, 1962). The results showed the transfer of male

sterility from the stock to the progenies of the scion.

This is of especial interest because male sterility

genes are carried by the mitochondria.

Non-Mendelian inheritance was also reported for

grafts of C. annuum by Kasahara and co-workers

(cited by Liu, 2006), with such data being confirmed on

the exact repetition of these experiments (Ohta &

Choung, 1975; Liu, 2006). Subsequently, using the

random amplified polymorphic DNA (RAPD) analysison grafts between C. baccatum and C. annuum,

Shiiguchi et al. (2004) demonstrated that the sequence

of the capsanthin-capsorubin synthase gene cloned

from the progeny was identical to that cloned from the

stock, although different from the scion in the Hap site.

This was interpreted as indicating that a transforma-

tional event could have occurred via the graft

(Shiiguchi et al., 2004). Zhang et al. (2002), working

with seedlings ofVigna radiata L grafted onto the stem

of Ipomea batatus L, also found that subsequent gen-

erations derived from seeds selfed on the scion had

distinct genetic variations but not in those from seeds

of the stock. Moreover, there was no restriction frag-ment length polymorphism in the cytoplasmic DNA

between the original scion and the variation. However,

a significant difference between the scion and variation

was recognized by the RAPD technique. There was no

evidence that indicated gene transformation from

stock to scion. Thus, preliminary evidence was

obtained indicating the transfer of genes via the graft

with expression in a subsequent generation.

As shown in Table 2, DNA (and RNA) could be

derived from a variety of sources in plants. It can be

expected that some nuclear material will be present

in the circulatory system through the loss of nuclei

from xylem vessels, tracheids, and phloem sieve ele-

ments on differentiation (Esau, 1953; Fahn, 1982).

Ohta (1991) reported that chromatin masses were

observed to move through the cell walls and inter-

cellular spaces from the lignifying cells of the stockand into the vascular system from where it was

suggested to move to the scion and thus cause

transformation in the fast-dividing scion floral

primordia.

It is possible that whole nuclei may be involved

because nuclei appear to be retained at a late stage in

xylem differentiation (Phillips & Dodds, 1977; Gahan

et al., 2003a). However, because apoptosis is not

involved in the eventual loss of such nuclei (Fukuda,

1996; Gahan et al., 2003a), it is unlikely that such

released whole nuclei will pass across the intact cell

walls of the apical meristem cells. It will require a

disintegration of such nuclei after release from thedifferentiating vascular cells to result in DNA frag-

ments that can enter the meristem cells.

DNA movement was found when cut shoots of

Lycopersicon esculentum were fed with 3H-thymidine,

re-rooted, and allowed to grow on. There was intense

DNA labelling in the collenchyma at the base of the

stem that was not a result of DNA synthesis in

preparation for mitosis. Over the subsequent 14

weeks, this label was slowly lost (Hurst, Gahan &

Snellen, 1973; Hurst & Gahan, 1975). Some of the

radioactive DNA leaving the collechymal nuclei of

tomato shoots at the base of the stem was found in

the apical meristem. This appeared to be a result ofthe movement of pieces of DNA in the plant and not

to breakdown products (nucleotides) that would have

been distributed throughout the plant and not be

detectable by autoradiography in any tissue. Prelimi-

nary results from similar experiments in which

shoots from nonradioactive plants were grafted

onto the stock of the plants fed with 3H-thymidine

showed occasional labelling of meristem cells of

the grafts (P. Gahan, unpubl. data). That DNA can

move throughout the plant has been demonstrated

both biochemically (Stroun et al., 1966, 1967) and

autoradiographically (Gahan, Anker & Stroun, 1973).

Moreover, when E. coli DNA carrying three markergenes (GUS, NPT II, and BAR genes) was fed to cut

shoots of Solanum aviculare and the stems re-rooted

and allowed to grow on, all three genes were found to

express throughout the plants. Importantly, the genes

were expressed also in the F1 generation, implying

that the genes had been integrated into chromosomes

of the apical meristem cells forming the floral tissues

(Gahan et al., 2003b). Hence, there is every reason to

suggest that DNA can move through the vascular

tissue from the stock to the graft as also proposed




10/18

by Stroun (1962) and Ohta (1991). By contrast to

the evidence of graft hybridization given above,

Stegmann & Bock (2009) showed that genetic mate-

rial is transferred between tobacco plants across graft

junctions. The exchange of transgenic markers was

demonstrated to occur in both directions, although

this involved the transfer of plastid DNA, rather thannuclear DNA, between cells. This was limited to the

site of the graft. The data obtained did not support

the concept of graft hybridization because it would be

analogous to sexual hybridization.

The passage of RNAs from the stock into scions has

been discussed in a review of the long distance trans-

port of RNA through phloem (Kehr & Buhtz, 2008).

Such RNAs can move through the phloem from the

stock plant to the scion where they could be detected

in the apical tissues, as well induce phenotypic

changes in the scions in some cases. Such RNA trans-

port was shown to occur with: (1) CmNACP mRNA

between pumpkin and cucumber (Ruiz-Medrano,Xoconostle-Czares & Lucas, 1999); (2) the transcript

of a KNOTTED1-like homeobox gene in tomato (Kim

et al., 2001); (3) BEL1-like transcription factor in

potato (Banerjee et al., 2006); and (4) GIBBERELLIC

ACID INSENSITIVE (GAI) gene in Cucurbita

maxima (Haywood et al., 2005). However, although

Arabidopsis GAI RNAs could traffic into non-

transgenic scion shoot apices and floral organs, they

were restricted from developing fruits, pedicels, and

peduncles (Haywood et al., 2005).

Hence, given the present information on RNA

movement from stock to scion, there is little evidence

of such RNAs inducing heritable changes in theprogeny of the scion. However, it is possible that

siRNA can pass from the stock to the scion because

such movement has been found across the junction

between semiparasitic Triphysaria and its host

(Tomilov et al., 2008). This is proposed to move

symplastically between the host and the parasite

(Vaughn, 2003). If this siRNA crosses the graft

junction, it would have to pass through any

plasmodesmata formed between the stock and scion.

It has been suggested that there are two major path-

ways for the movement of siRNA: locally via the

plasmodesmata and long distance via the phloem

(Melnyk et al., 2011). Because no RNA is normallyfound in the xylem flow, it is difficult to accept that

the siRNA in the recipient plant could act other than

locally because the siRNA is unlikely to move

upwards via the phloem transport system.

PARASITIC/EPIPHYTIC PLANTS/HOSTGENE MOVEMENT

Clearly, the mechanism by which circulating nucleic

acids could move to the progeny of an individual plant

is ever-present. However, grafting is a man-made

process and is very unlikely to occur in the wild.

Nevertheless, there are attachments found between

host and parasitic plants that may be considered to be

similar to grafts in that they could permit gene

exchanges between the host and parasite in both

directions. Indeed, Bergthorsson et al. (2003, 2004)and Bock (2010) suggested that HGT is particularly

prevalent between organisms that are either inti-

mately associated or establish, at least occasionally,

cellcell contacts (i.e. in either an epiphytic or a

parasitic relationship). In line with this approach is

the HGT of part of the mitochondrial genome of the

endoparasitic plant, with Rafflesia being acquired

from their obligate host Tetrastigma (Davis &

Wurdack, 2004), whereas part of the mitochondrial

genome of Phaseolus appears to have come from the

chloroplast of an, as yet, unidentified non-eudicot

plant (Woloszynska et al., 2004).

Bergthorsson et al. (2004) have listed some twentygenes found in the angiosperm Amborella that have

been derived primarily from eudicots and bryophytes.

Species of the parasitic broomrape genus Phelipanche

have two copies of the plastid ribosomal gene rps2,

one of which corresponds to a gene, apparently hori-

zontally acquired, from the related broomrape genus

Orobanche. Because the donor and the recipient

plants are both parasitic plants, it is possible that the

exchange of genetic material could have occurred via

a common host (Park et al., 2007). Similarly, three

species of plantain have a normally functioning copy

of the mitochondrial apt1 gene together with a second

defective copy that strongly resembles the apt1 genepresent in Cuscuta (Mower et al., 2010). Phloem-

mobile mRNAs are also considered to traffic from the

host, Lycopersicon esculentum Mill., to the plant para-

site Cuscuta pentagona Engelm (Roney, Khatibi &

Westwood, 2007).

In addition, there has been HGT of a gene of

unknown function from Sorghum bicolor to the

nucleus of the parasitic plant Striga hermonthica

(Yoshida et al., 2010). There are also indications

that two mitochondrial gene regions of the fern

Botrychium virginianum (L.) Sw. may have originated

from the root-parasitic Loranthaceae possibly by HGT

(Davis et al., 2005).An hemiparasitic plant such as Triphysaria

has been shown to take up siRNA from its host

plant (e.g. lettuce) (Tomilov et al., 2008), probably

symplastically between the two species (Vaughn,

2003).

Thus, the CNAs present in vascular plants appear

to include genes from parasitic and epihytic plants so

presenting HGT via a relatively simple mechanism

of circulation and incorporation into vegetative cells

predetermined to form gametes.

10 P. GAHAN



11/18

RELEASE AND UPTAKE OF DNABY PLANT MITOCHONDRIA

Many DNA sequences appear to be exchanged

between plant mitochondria of especially parasitic

plants and their hosts. Although little is known about

the release of DNA from plant mitochondria, otherthan through cell death, the uptake of DNA into plant

mitochondria has been well studied.

In higher plants, especially angiosperms, mito-

chondrial genomes tend to be much larger

(300800 kb) than the DNA of animal or fungal mito-

chondria (1685 kb) yet only 1118% corresponds to

protein or structural RNA genes, whereas 5% or more

appears to have originated from plastids, nuclei or

viruses; probably approximately half has no recogniz-

able function and origin (Marienfeld, Unseld &

Brennicke, 1999; Kubo et al., 2000). Koulintchenko,

Konstantinov & Dietrich (2003) have demonstrated a

mechanism for DNA entry into plant mitochondria.They proposed the existence of an active, trans-

membrane potential-dependent mechanism of DNA

uptake to be involved. They showed that, although

the process was restricted to dsDNA, there was no

obvious sequence specificity, the most efficient uptake

occurring with linear fragments up to a few kilobase

pairs in size. The uptake process appears to be active

and to involve a voltage-dependent anion channel

with an adenine nucleotide translocator. These repre-

sent the core components of the animal mitochondrial

permeability transition pore complex associated with

the inner mitochondrial membrane (see above).

However, there is no reliance on known mitochondrialmembrane permeabilization processes. Interestingly,

the imported DNA was transcribed. The uptake

mechanism proposed by Koulintchenko, Konstantinov

& Dietrich (2003) could explain the facility of the

simultaneous transfer of three mitochondrial genes,

atp1, atp6 and matR, from the Cuscuta gronovii

genome to that of the host Plantago coronopus

mitochondrial genome in the form of one piece of

DNA (Mower et al., 2010). In addition, based on the

data concerning the easy mobility of mitochondrial

DNA fragments between parasitic plants and their

hosts, Hao et al. (2010) have proposed a model in

which intra- and inter-organellar gene transfer/recombination are important in the generation of

mitochondrial genetic diversity. Archibald & Richards

(2010) have discussed the impact and implications of

such a model. However, the modified mitochondria

would need to be present in plant egg cells to have an

impact on the F1 generation.

The mitochondrial permeability transition pore

complex exists in animal mitochondria where it has

been well studied (Bernardi, 2013). Although it

permits the passage of molecules of up to 1500 Da,

currently, there is no evidence for a similar passage of

DNA to that seen with plant mitochondria.

PLANT PATHOGENS

Plants are subject to many pathogen attacks, with the

major groups involving viruses, bacteria and fungi, aswell as insects and nematodes. At present, although

there is little evidence of HGT from prokaryotes to

plants as a result, perhaps, of there being little work

in this field (Keeling & Palmer, 2008), genes for

multiple enzymes for carotenoid biosynthesis appear-

ing to have been transferred from fungi to aphids

(Moran & Jarvik, (2010) and tobacco to bacteria

(Pontoroli et al. 2009; Demanche et al., 2011). In

addition, there are few reports of HGT from

the pathogens to the host plants (Nielsen et al.,

1998), again, because this field has not been fully

investigated.

It is also possible for the spread of RNA silencingfrom a plant to an invertebrate and fungal pathogens

(Huang et al., 2006; Baum et al., 2007; Mao et al.,

2007; Nowara et al., 2010; Tinoco et al., 2010). The

assumption has been made that siRNAs are trans-

ferred between organisms, although it cannot be

ruled out that siRNA precursors are the mobile form

that is subsequently processed in the host. When

sucking insects and nematodes feed, the cells are

broken so that active siRNA is involved (Melnyk

et al., 2011). This appears to be the case for

Coleoptera when the RNA silencing signal remains

functional.

As is the case with the animal systems, the mecha-nisms by which CNAs in plants can integrate into the

germ cells is present and there are examples to show

that such nucleic acids can pass to the F1 generations.

Furthermore, not only is there is evidence for HGT

from prokaryotes to eukaryotic plants, but also there

is some evidence for HGT between eukaryotic plants.

Hence, not only are the potential mechanisms for

gene movement and inheritance both present in

plants, but also preliminary studies indicate that

CNAs in plants could result in an impact on plant

evolution.

Thus, the preliminary studies of HGT (1) between

bacteria and plant parasitic nematodes showing thetransfer of genes involved with carbohydrate metabo-

lism to improve the nematodes plant invasive prop-

erties (Scholl et al., 2003) and (2) between transgenic

plants and soil bacteria (Nielsen et al., 1998), as well

as (3) the natural movement of genes between differ-

ent plant species and, for example, prokaryotes and

fungi into plants, can occur by gene transfer medi-

ated by, for example, micro-organisms, parasites,

viruses or mites and by direct cell-to-cell transfer at

a relatively low frequency. However, this frequency




12/18

may be sufficiently high to act as a significant factor

in the genetic changes of a chromosome during evo-

lutionary time (Richardson & Palmer, 2007; Syvanen,

2012). Interestingly, HGT in plants appears to

involve a comparatively high level of exchange

between mitochondria. It should not be forgotten

that, in plants, the horizontal transfer of mobile ele-ments of either DNA or RNA, either as a naked

nucleic acid or within a virus, could occur especially

through, for example, tissues damaged by insects.

Such foreign mobile elements could then be taken up

and incorporated into the plant genomes. However, in

multicellular organisms, it is likely that such a trans-

fer would rarely occur directly into germ cell tissues

and so be transmitted to the next generation

(Bennetzen, 2000). Nevertheless, as described above,

mobile DNA can readily enter vegetative cells of the

shoot apex prior to forming the reproductive organs

and so appear in the F1 generation (Gahan et al.,

2003b).

CONCLUSIONS

Both animal and vascular plant systems have the

capacity for CNAs to enter the reproductive systems

where, in vascular plants, they are seen to influence

the F1 generation. However, in eutherian systems,

although there is a possible mechanism by which

CNAs can influence the F1 generation, currently,

there are few examples regarding the success of

HGT and the uptake of CNAs entering the F 1 gen-

eration. Such examples appear to be restricted tolower organisms such as bdelloid rotifers (Boschetti

et al., 2012) or Lepidoptera (Sun et al., 2013). This

may be a result, in part, of the limited number of

observations made. It also appears to be influenced

by the germ cell line leading to the formation of

gametes and to the way in which modified gametes

are made available that may well limit the possi-

bilities of the F1 being modified. By contrast, the

vegetative cells of vascular plants are more readily

available to genetic modification prior to producing

reproductive elements. This process also allows

genetic fragments transferred by HGT not only to

enter somatic cells, but, more importantly, alsooffers an explanation for the successful entry into

gametes and hence the continuation in the subse-

quent generation(s) of the recipient vascular plant,

although this is less so in higher animals. Clearly,

there is a requirement for a more systematic study

of CNAs and HGT in vascular plant systems, and

especially of CNAs in animal systems, with the aim

of further clarifying a possible influence of CNAs on

F1 and subsequent generations, as well as in the

evolutionary process.

ACKNOWLEDGEMENTS

I wish to thank the reviewers for their very helpful

comments for the improvement of the manuscript and

to Dr Maurice Stroun and Professor M. S. Black for

stimulating discussion.

REFERENCES

Adams DH, Diaz N, Gahan PB. 1997. In vitro stimulation

by tumour cell media of [3H]thymidine incorporation by

mouse spleen lymphocytes. Cell Biochemistry & Function

15: 119126.

Adams DH, Macintosh AAG. 1985. Studies on the cytosolic

DNA of chick embryo fibroblasts and its uptake by recipient

cultured cells. International Journal of Biochemistry 17:

10411051.

Anderson RG, Kamen BA, Rothberg KG, Lacey SW.

1992. Potocytosis: sequestration and transport of small mol-

ecules by caveolae. Science 255: 410411.

Anker P, Jachertz D, Maurice PA, Stroun M. 1984. Nude

mice injected with DNA released by antigen stimulated

human T lymphocytes produce specific antibodies express-

ing human characteristics. Cell Biochemistry & Function 2:

3337.

Anker P, Jachertz D, Stroun M, Brogger R, Lederrey C,

Henri J, Maurice P. 1980. The role of extracellular DNA

in the transfer of information from T to B human lympho-

cytes in the course of an immune response. Journal of

Immunogenetics 6: 475481.

Anker P, Lyautey J, Lefort F, Lederrey C, Stroun M.

1994. Transformation of NIH/3T3 cells and SW 480 cells

displaying K-ras mutation. Comptes Rendues de l Academie

des Sciences III 10: 869874.

Archibald JM, Richards TA. 2010. Gene transfer:anything goes in plant mitochondria. BMC Biology 8: 147

149.

Balicki D, Beutler E. 1997. Histone H2A significantly

enhances in vitro DNA transfection. Molecular Medicine 3:

782787.

Balicki D, Putnam CD, Scaria PV, Beutler E. 2002.

Structure and function correlation in histone H2A peptide-

mediated gene transfer. Proceedings of the National

Academy of Sciences of the United States of America 99:

74677471.

Balicki D, Reisfeld RA, Pertl U, Beutler E, Lode HN.

2000. Histone H2A-mediated transient cytokine gene deliv-

ery induces efficient antitumor responses in murine neurob-

lastoma. Proceedings of the National Academy of Sciences of

the United States of America 97: 1150011504.

Banerjee AK, Chatterjee M, Yu Y, Suh S-G, Allen W,

Miller A, Hannapel DJ. 2006. Dynamics of a mobile RNA

of potato involved in a long-distance signaling pathway. The

Plant Cell 18: 34433457.

Barkman TJ, McNeal JR, Lim S-H, Coat G, Croom HB,

Young ND, de Pamphilis CW. 2007. Mitochondrial DNA

suggests at least 11 origins of parasitism in angiosperms

and reveals genomic chimerism in parasitic plants. BMC

Evolutionary Biology 7: 248.

12 P. GAHAN



13/18

Basner-Tschakarjan E, Mirmohammadsadegh A, Baer

A, Hengge UR. 2004. Uptake and trafficking of DNA in

keratinocytes: evidence for DNA-binding proteins. Gene

Therapy 11: 765774.

Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P,

Ilagan O, Johnson S, Plaetinck G, Munyikwa T, Pleau

M, Vaughn T, Roberts J. 2007. Control of coleopteran

insect pests through RNA interference. Nature Biotechnol-

ogy 25: 13221326.

Beck J, Urnovitz HB, Riggert J, Clerici M, Schtz E.

2009. Profile of the circulating DNA in apparently healthy

individuals. Clinical Chemistry 55: 730738.

Bennetzen JL. 2000. Transposable element contributions to

plant gene and genome evolution. Plant Molecular Biology

42: 251269.

Benoit J, Leroy P, Vendrely R, Vendrely C. 1960. Experi-

ments on Pekin ducks treated with DNA from Khaki Camp-

bell ducks. Transactions of the New York Academy of Science

22: 494503.

Benoit J, Leroy P, Vendrely R, Vendrely C. 1966. Heredi-

tary modifications of morphological characteristics in thePekin duck following the injection of DNA from the Khaki

Campbell duck, 13th Worlds Poultry Congress, Kiev, 100

105.

Bergsmedh A, Szeles A, Henriksson M, Bratt A, Folkma

MJ, Spetz AL, Holmgren L. 2001. Horizontal transfer of

oncogenes by uptake of apoptotic bodies. Proceedings of the

National Academy of Sciences of the United States of

America 98: 64076411.

Bergthorsson U, Adams KL, Thomason B, Palmer JD.

2003. Widespread horizontal transfer of mitochondrial

genes in flowering plants. Nature 424: 197201.

Bergthorsson U, Richardson A, Gergory J, Young GJ,

Leslie R, Goertzen LR, Palmer D. 2004. Massive hori-

zontal transfer of mitochondrial genes from diverse land

plant donors to the basal angiosperm Amborella. Proceed-

ings of the National Academy of Sciences of the United

States of America 101: 1774717752.

Bernardi P. 2013. The mitochondrial permeability transition

pore: a mystery solved? Frontiers in Physiology 4: 95116.

Bock R. 2010. The give-and-take of DNA: horizontal gene

transfer in plants. Plant Science 15: 1122.

Boriachok-Nizhnik GV. 1951. Experiments in vegetative

hybridization of animals. Zhurnal Obshchei Biologii 12:

233251.

Boschetti C, Carr A, Crisp A, Eyres I, Wang-Koh Y,

Lubzens E, Barraclough TG, Micklem G, Tunnacliffe

A. 2012. Biochemical diversification through foreign geneexpression in Bdelloid Rotifers. PLoS Genetics 8: e1003035.

doi:10.1371/journal.pgen.1003035

Bottger M, Zaitsev SV, Otto A, Haberland A, Vorobev

VI. 1998. Acid nuclear extracts as mediators of gene trans-

fer and expression. Biophysica Acta 1395: 7887.

Brackett BG, Baranska W, Sawicki W, Koprowski H.

1971. Uptake of heterologous genome by mammalian sper-

matozoa and its transfer to ova through fertilization. Pro-

ceedings of the National Academy of Sciences of the United

States of America 68: 353357.

Brindley PJ, Mitreva M, Ghedin E, Lustigman S. 2009.

Helminth genomics: the implications for human health.

PLoS Neglected Tropical Diseases 3: e538.

Budker V, Hagstrom JE, Lapina O, Eifrig D, Fritz J,

Wolff JA. 1997. Protein/amphipathic polyamine complexes

enable highly efficient transfection with minimal toxicity.

BioTechniques 23: 142147.

Bulicheva N, Fidelina O, Krtumova MN, Neverova M,

Bogush A, Bogush M, Roginko O, Veiko N. 2008. Effect

of cell-free DNA of patients with cardiomyopathy and rDNA

on the frequency of contraction of electrically paced neona-

tal rat ventricular myocytes in culture. Annals of the New

York Academy of Science 1137: 273277.

Burbank L. 19141915. His methods and discoveries and

their practical application, 12 volumes. New York, NY:

Luther Burbank Press.

Carballada R, Esponada P. 2001. Regulation of foreign

DNA uptake by mouse spermatozoa. Experimental Cell

Research 262: 104113.

Chang K-T, Ikeda A, Hayashi K, Furuhata Y, Bannai M,

Nishihara M, Ohta A, Ogawa S, Takahashi M. 1999.Possible mechanisms for the testis-mediated gene transfer

as a new method for producing transgenic animals. Journal

of Reproduction and Development 45: 3742.

Craig JP, Bekal S, Hudson M, Domier L, Niblack T,

Lambert KN. 2008. Analysis of a horizontally transferred

pathway involved in vitamin B6 biosynthesis from the

soybean cyst nematode heterodera glycines. Molecular

Biology & Evolution 25: 20852098.

Dalpke A, Frank J, Peter M, Heeg K. 2006. Activation of

toll-like receptor 9 by DNA from different bacterial species.

Infection and Immunity 74: 940946.

Darwin C. 1868. The variation of animals and plants under

domestication, two volumes. London: John Murray.

Davis BD, Dulbecco R, Eisen HN, Ginsberg HS, Wood

WB. 1973. Microbiology, 2nd edn. Hagerstown, MD: Harper

and Row.

Davis CC, Anderson WR, Wurdack KJ. 2005. Gene trans-

fer from a parasitic flowering plant to a fern. Proceedings of

the Royal Society of London Series B, Biological Sciences

272: 22372242.

Davis CC, Wurdack KJ. 2004. Host-to-parasitic gene trans-

fer in flowering plants. Science 305: 676678.

Demanche S, Monier J-M, Dugat-Bony E, Simonet P.

2011. Exploration of horizontal gene transfer between

transplastomic tobacco and plant-associated bacteria.

FEMS Microbial Ecology 78: 129136.

Demirhan I, Hasselmayer O, Chandra A, Ehemann M,Chandra P. 1998. Histone-mediated transfer and expres-

sion of the HIV-1 tat gene in Jurkat cells. Journal of Human

Virology 1: 430440.

Doolittle RF, Feng DF, Anderson KL, Alberro MR. 1990.

A naturally occurring horizontal gene transfer from a

eukaryote to a prokaryote. Journal of Molecular Evolution

31: 383388.

Dunning Hotopp JC, Clark ME, Oliveira DCSG, Foster

JM, Fischer P, Muoz Torres MC, Giebel JD, Kumar

N, Ishmael N, Wang S, Ingram J, Nene RV, Shepard J,




14/18

Tomkins J, Richards S, Spiro DJ, Ghedin E, Slatko

BE, Tettelin H, Werren JH. 2007. Widespread lateral

gene transfer from intracellular bacteria to multicellular

eukaryotes. Science 317: 17531755.

Dworetzky SJ, Lanford RE, Feldherr CM. 1988. The

effects of variations in the number and sequence of target-

ing signals on nuclear uptake. Journal of Cell Biology 107:

12791287.

Ermakov AV, Kostyuk SV, Konkova MS, Egolina NA,

Malinovskaya EM, Veiko NN. 2008. Extracellular DNA

fragments. Factors of stress signaling between X-irradiated

and non-irradiated human lymphocytes. Annals of the New

York Academy of Science 1137: 4146.

Esau K. 1953. Plant anatomy. New York NY: Wiley & Sons

Inc.

Fahn A. 1982. Plant anatomy, 3rd edn. Oxford: Pergamon

Press.

Frankel R. 1954. Graft-induced transmission to progeny of

cytoplasmic male sterility in petunia. Science 124: 684

685.

Frankel R. 1962. Future evidence on graft induced transmis-sion to progeny of cytoplasmic male sterility in petunia.

Genetics 47: 641646.

Fritz JD, Herweijer H, Zhang G, Wolff JA. 1996. Gene

transfer into mammalian cells using histone-condensed

plasmid DNA.Hum. Gene Therapy 7: 13951404.

Fukuda H. 1996. Xylogenesis: initiation, progression and cell

death. Annual Revue of Plant Physiology & Plant Molecular

Biology 47: 299325.

Gahan PB. 2012. Biology of circulating nucleic acids and

possible roles in diagnosis and treatment of diabetes and

cancer. Infectious Disorders Drug Targets 12: 360370.

Gahan PB. 2013. Circulating nucleic acids in plasma and

serum: applications in diagnostic techniques for noninvasive

prenatal diagnosis. International Journal of Womens

Health 5: 177186.

Gahan PB, Anker P, Stroun M. 1973. An autoradiographic

study of bacterial DNA in Lycopersicum esculentum. Annals

of Botany 37: 681685.

Gahan PB, Anker P, Stroun M, Jacob K. 1968. DNA-

induced chromosome damage in Vicia faba. Caryologia 22:

307310.

Gahan PB, Perry JI, Stroun M, Anker P. 1974. Effect of

exogenous DNA on acid deoxyribonuclease activity in intact

roots of Vicia faba L. Annals of Botany 38: 222226.

Gahan PB, Stroun M. 2010a. The biology of circulating

nucleic acids in plasma and serum. In: Rykova EY, Kikuchi

Y, eds. Extracellular nucleic acids. NAMB series: NucleicAcids and Molecular Biology. Berlin: Springer.

Gahan PB, Stroun M. 2010b. The virtosome a novel

cytosolic informative entity and intercellular messenger.

Cell Biochemistry & Function 28: 110.

Gahan PB, Wang L, Bowen ID, Winters C. 2003a.

Cytokinin-induced apoptotic nuclear changes in cotyledons

of Solanum aviculare and Lycopersicon esculentum. Plant

Cell Tissue and Organ Culture 72: 213221.

Gahan PB, Wyndaele R, Mantell SH, Baggetti B. 2003b.

Evidence that direct DNA uptake through cut shoots leads

to genetic transformation of Solanum aviculare Forst. Cell

Biochemistry Function 21: 1117.

Garcia-Olmo D, Garcia-Olmo DC, Domnguez-Berzosa

C, Guadalajara H, Luz Vega L, Garca-Arranz M. 2012.

Oncogenic transformation induced by cell-free nucleic acids

circulating in plasma (genometastasis) remains after the

surgical resection of the primary tumor: a pilot study.

Expert Opinion in Biological Therapeutics 12: S61S68.

Garcia-Olmo DC, Dominguez C, Garcia-Arranz M,

Anker P, Stroun M, Garca-Verdugo JM, Garca-Olmo

D. 2010. Cell-free nucleic acids circulating in the plasma of

colorectal cancer patients induce the oncogenic transforma-

tion of susceptible cultured cells. Cancer Research 70: 560

567.

Garcia-Olmo DC, Ruiz-Piueras R, Garcia-Olmo D. 2004.

Circulating nucleic acids in plasma and serum (CNAPS) and

its relation to stem cells and cancer, metastasis: state of the

issue. Histology & Histopathology 19: 575583.

Garca-Olmo D, Garca-Olmo DC, Ontan J, Martnez

E. 2000. Horizontal transfer of DNA and the

genometastasis hypothesis. Blood 95: 724725.Garca-Olmo D, Garca-Olmo DC, Ontan J, Martnez

E, Vallejo M. 1999. Tumor DNA circulating in the plasma

might play a role in metastasis. The hypothesis of

genometastasis. Histology & Histopathology 14: 11591164.

Gibbings D, Voinnet O. 2010. Control of RNA silencing and

localization by endolysosomes. Trends in Cell Biology 20:

491501.

Gilbert CG, Schaack S, Pace JK II, Brindly P, Feshotte

C. 2010. A role for hostparasite interactions in the hori-

zontal transfer of transposons across phyla. Nature 464:

13471350.

Giordano R, Magnano AR, Zaccagnini G, Pittoggi C,

Moscufo N, Lorenzini R, Spadofora C. 2000. Reverse

transcriptase activity in mature spermatozoa of mouse.

Journal of Cell Biology 148: 11071114.

Gladishev EA, Meselson M, Arkhipova IR. 2008. Massive

horizontal gene transfer in bdelloid rotifers. Science 320:

12101213.

Glouchtchenko IE. 1948. Vegetative hybridization in

massive horizontal gene transfer in plants [in Russian].

Moscow: Academy Nauk SSR.

Guang S, Bochner AF, Pavelec DM, Burkhart KB,

Harding S, Lachowiec J, Kennedy S. 2008. An

Argonaute transports siRNAs from the cytoplasm to the

nucleus. Science 321: 537541.

Guerra R, Carballada R, Esponda P. 2005. Transfection of

spermatozoa in bivalve molluscs using naked DNA. CellBiology International 29: 159164.

Haberland A, Knaus T, Zaitsev SV, Buchberger B, Lun

A, Haller H, Bottger M. 2000. Histone H1-mediated

transfection: serum inhibition can be overcome by Ca2+

ions. Pharmaceutical Research 17: 229235.

Haegle H, Allam R, Pawar RD, Anders J-H. 2009. Double-

stranded RNA activates type I interferon secretion in

glomerular endothelial cells via retinoic acid-inducible gene

(RIG)-1. Nephrology Dialysis Transplantation 24: 3312

3318.

14 P. GAHAN



15/18

Hamada N, Matsumoto H, Hara T, Kobayashi Y. 2007.

Intercellular and intracellular signaling pathways mediat-

ing ionizing radiation-induced bystander effects. Journal of

Radiation Research 48: 8795.

Hao W, Richardson AO, Zheng Y, Palmer JD. 2010.

Gorgeous mosaic of mitochondrial genes created by horizon-

tal transfer and gene conversion. Proceedings of the

National Academy of Sciences of the United States of

America 107: 2157621581.

Hariton-Gazal E, Rosenbluh J, Graessmann A, Gilon C,

Loyter A. 2003. Direct translocation of histone molecules

across cell membranes. Journal of Cell Science 116: 4577

4586.

Haywood V, Yu T-S, Huang N-C, Lucas WJ. 2005.

Phloem long-distance trafficking of GIBBERELLIC ACID-

INSENSITIVE RNA regulates leaf development. The Plant

Journal 42: 4968.

Hei TK, Zhou H, Ivanov VN, Hong M, Lieberman HB,

Brenner DJ, Amundson SA, Geard CR. 2008. Mecha-

nism of radiation-induced bystander effects: a unifying

model. Journal of Pharmacy & Pharmacology 60: 943950.Heinemann JA, Sprague GF. 1989. Bacterial conjugative

plasmids mobilize DNA transfer between bacteria and

yeast. Nature 340: 205209.

Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo

H, Matsumoto M, Hoshino K, Wagner H, Takeda K,

Akira S. 2002. A Toll-like receptor recognizes bacterial

DNA. Journal of Leukocyte Biology 71: 538544.

Hemmi H, Takeuchi O, Kawai T, Kaisho TS, Sanjo H,

Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira

SA. 2000. Toll-like receptor recognizes bacterial DNA.

Nature 408: 740745.

Hirata Y. 1979. Graft-induced changes in eggplant (S.

melongena L.) I. Changes of hypocotyl color in the grafted

scions and in the progenies of the grafted scions. Japanese

Journal of Breeding 29: 318323.


melongena L.) II . Changes of fruit color and fruit shape in

the grafted scions and in the progenies of the grafted scions.

Japanese Journal of Breeding 30: 8390.


melongena L.) I. Appearance of the changes. Euphytica 35:

395401.

Hirata Y, Noguchi T, Oguni S, Kan T. 2001. Induction of

male sterility in the progeny derived from interspecific

chaemera between Brassica oleracea and B. campestris.

Euphytica 117: 143149.

Hirata Y, Ogata S, Kurita S, Nozawa GT, Zhou J, Wu S.2003. Molecular mechanism of graft transformation in Cap-

sicum annuum L. Acta Horticultura 625: 125130.

Holmgren L, Szeles A, Rajnavolgyi E, Folkman J,

Klein G, Ernberg I, Falk KI. 1999. Horizontal transfer of

DNA by the uptake of apoptotic bodies. Blood 93: 3956

3963.

Huang G, Allen R, Davis EL, Baum TJ, Hussey RS. 2006.

Engineering broad root-knot resistance in transgenic plants

by RNAi silencing of a conserved and essential root-knot

nematode parasitism gene. Proceedings of the National

Academy of Sciences of the United States of America 103:

1430214306.

Hurst PR, Gahan PB. 1975. Turnover of DNA in ageing

tissues of Lysopersicon esulentum. Annals of Botany 39:

7176.

Hurst PR, Gahan PB, Snellen JW. 1973. Turnover of

labelled DNA in differentiated collenchyma. Differentiation

1: 261266.

Jans DA, Hubner S. 1996. Regulation of protein transport to

the nucleus: central role in phosphorylation. Physiological

Reviews 76: 651685.

Johnson J, Canning J, Kaneko T, Pru JK, Tilly JL. 2004.

Germline stem cells and follicular renewal in the postnatal

mammalian ovary. Nature 428: 145150.

Keeling PJ, Palmer JD. 2008. Horizontal gene transfer in

eukaryotic evolution. Nature Reviews Genetics 9: 605618.

Kehr J, Buhtz A. 2008. Long distance transport and move-

ment of RNA through the phloem. Journal of Experimental

Botany 59: 8592.

Kim M, Canio W, Kessler S, Sinha N. 2001. Developmental

changes due to long-distance movement of a homeoboxfusion transcript in tomato. Science 293: 287289.

Kiss A. 2012. Caveolae and the regulation of endocytosis.

OGY Advances in Experimental Medicine & Biology 729:

1428.

Kiss AL, Botos E. 2009. Compartments to avoid lysosomal

degradation? Journal of Cellular and Molecular Medicine

13: 1122811237.

Klokov D, Criswell T, Leskov KS, Araki S, Mayo L,

Boothman DA. 2004. IR-inducible clusterin gene expres-

sion: a protein with potential roles in ionizing radiation-

induced adaptive responses, genomic instability, and

bystander effects. Mutatation Research 568: 97110.

Koulintchenko M, Konstantinov Y, Dietrich A. 2003.

Plant mitochondria actively import DNA via the permeabil-

ity transition pore complex. EMBO Journal 22: 1245

1254.

Kubo T, Nishizawa S, Sugawara A, Itchoda N, Estiati A,

Mikami T. 2000. The complete nucleotide sequence of the

mitochondrial genome of sugar beet (Beta vulgaris L.)

reveals a novel gene for tRNA(Cys)(GCA). Nucleic Acids

Research 28: 25712576.

Lanes CFC, Sampaio LA, Marins LF. 2009. Evaluation of

DNase activity in seminal plasma and uptake of exogenous

DNA by spermatozoa of the Brazilian flounder Paralichthys

orbignyanus. Theriogenology 71: 525533.

Ledoux L, Charles P. 1970. Fate of exogenous DNA in

mammals. In: Ledoux L, ed. Uptake of informative moleculesby living cells. Amsterdam: North-Holland Publishing

Company, 397413.

Lee YS, Pressman S, Andress AP, Kim K, White JL,

Cassidy JJ, Li X, Lubel K, Lim H, Cho IS et al. 2009.

Silencing by small RNAs is linked to endosomal trafficking.

Nature Cell Biology 11: 11501156.

Liu Y-S. 2006. The historical and modern genetics of plant

graft hybridization. Advances in Genetics 56: 101129.

Lorimore SA, Wright EG. 2003. Radiation-induced genomic

instability and bystander effects: related inflammatory-type




16/18

responses to radiation-induced stress and injury? Interna-

tional Journal of Radiation Biology 79: 1525.

Luo D, Saltzman WM. 2000. Synthetic DNA delivery. Nature

Biotechnology 18: 3337.

Mao YB, Cai WJ, Wang JW, Hong GJ, Tao XY, Wang LJ,

Huang YP, Chen XY. 2007. Silencing a cotton bollworm

P450 monooxygenase gene by plant-mediated RNAi impairs

larval tolerance of gossypol. Nature Biotechnology 25: 1307

13

Documents

Gahan 2013 Acidos Nucleicos Efectos Inherencia