HLA-C “blank” alleles express class I gene products. Biochemical analysis of four different HLA-C “blank” polypeptides

Monoclonal antibodies (mAbs) W6/32, HC10, and 4E were used to precipitate class I antigens from 21 selected individuals with at least oneHLA-C “blank” allele. In 19 of these individuals, characteristicHLA-C banding patterns which could be precipitated by all three HLA class I mAbs were observed on one-dimensional isoelectric focusing gels-obviously the gene products ofHLA-C “blank”. At least four allelic HLA-C “blank” gene products with different isoelectric points could be discerned. All of them segregated withHLA-C “blank” haplotypes in informative families; two of them were associated withHLA-B51, one withHLA-B38, and one withHLA-B18. Reactivity of the HLA-C “blank” heavy chains with mAb W6/32 indicates that they are able to associated with beta-2 microglobulin, and hence are most probably expressed at the cell surface.     

Extended MHC haplotypes and CYP21/C4 gene organisation in Irish 21-hydroxylase deficiency families

Summary We have analysed fifteen classical 21-hydroxylase deficiency families from throughout Southern Ireland and report the serologically defined HLA-A, HLA-B, HLA-Cw, HLA-DR, C4A and C4B polymorphisms that characterize the inferred disease haplotypes. Additionally, we have used a combination of short and long range restriction mapping procedures in order to characterize the CYP21/C4 gene organization associated with individual serologically defined haplotypes. The results obtained indicate that disease haplotypes are characterized by a high frequency (33%) of CYP21B gene deletion and 8 out of 10 such deletion haplotypes are represented by the extended haplotype HLA-DR1, C4BQo, C4A3, HLA-B40(w60), HLA-Cw3, HLA-A3. Large scale length polymorphism in the CYP21/C4 gene cluster was found to conform strictly to a variable number of tandem repeats model with 4 alleles being detected. Disease haplotypes in which defective CYP21B gene expression is inferred to result from pathological point mutations show extensive diversity of associated HLA markers and include two examples of the extended HLA haplotype HLA-DR3, B8, Cw7, A1 haplotype, which has previously been reported to be negatively associated with 21-hydroxylase deficiency. One unusual disease haplotype has two CYP21 + C4 units, both of which appear to contain CYP21B-like genes.     

Null alleles of the fourth component of complement and HLA haplotypes in familial systemic lupus erythematosus

Eight families (121 individuals) with two or more members affected with systemic lupus erythematosus (SLE) were analyzed for histocompatibility antigens (HLA-A, B, C, DR, MT, and MB) and complement antigens (C4A, C4B, and BF). These data were correlated with serological markers (antinuclear antibodies, single- and double-stranded anti-DNA, anti-SM, anti-nRNP, anti-Ro [SS-A], anti-La [SS-B], and biological false-positive tests for syphilis and clinical features. Fifteen members had SLE, and 19 had other immune diseases (subacute cutaneous lupus erythematosus, discoid lupus erythematosus, hypothyroidism, insulin-dependent diabetes mellitus, primary, Sjogren's syndrome, immune thrombocytopenic purpura, rheumatoid arthritis, and multiple sclerosis). Twenty-three healthy relatives (seroreactors) had significant titers of circulating antibodies, as did 2 of 17 spouses. There was an increased frequency of null C4 alleles in those individuals with SLE (60%) and healthy relatives (50%) as compared with spouses (24%). Multivariate analysis showed a significant association between SLE and female sex (P=.006), whereas there was no significant association revealed between female sex and other immune diseases. Patients with SLE also had a higher frequency of either C4A or C4B null alleles (P=.01) than those with immune diseases. The C4A homozygous null phenotype was more common in SLE patients than in seroreactors (P=.02). There was a higher frequency of HLA-DR2 and DR3 in individuals with SLE than in those with immune disease (P=.08), seroreactors (P=.02) and normal relatives (P =.002). One totally C4-deficient patient with SLE was identified. These families demonstrate an important association between SLE and the C4 null allele and the HLA-DR2 and DR3. These risk factors, however, cannot account for the development of disease in all individuals.     

The “Sardinian”HLA-A30,B18,DR3,DQw2 haplotype constantly lacks the21-OHA andC4B genes. Is it an ancestral haplotype without duplication?

TheC4 and21-OH loci of the class III HLA have been studied by specific DNA probes and the restriction enzymeTaq I in 24 unrelated Sardinian individuals selected from completely HLA-typed families. All 24 individuals had theHLA extended haplotypeA30,Cw5,B18, BfF1,DR3,DRw52,DQw2, named “Sardinian” in the present paper because of its frquency of 15% in the Sardinian population. Eighteen of these were homozygous for the entire haplotype, and six were heterozygous at theA locus and blank (or homozygous) at all the other loci. In all completely homozygous cells and in four heterozygous cells at theA locus, the restriction fragments of the21-OHA (3.2 kb) andC4B (5.8 kb or 5.4 kb) genes were absent, and the fragments of theC4A (7.0 kb) and21-OHB (3.7 kb) genes were present. It is suggested that the “Sardinian” haplotype is an ancestral haplotype without duplication of theC4 and21-OH genes, practically always identical in its structure, also in unrelated individuals. The diversity of this haplotype in the class III region (about 30 kb less) may be at least partially responsible for its misalignment with most haplotypes, which have duplicatedC4 and21-OH genes, and therefore also for its decreased probability to recombine. This can help explain its high stability and frequency in the Sardinian population. The same conclusion can be suggested for the Caucasian extended haplotypeA1,B8,DR3 that always seems to lack theC4A and21-OHA genes.     

Activities of human recombinant cytochrome P450 isoforms and human hepatic microsomes for the hydroxylation ofAlternaria toxins

TheAlternaria toxins alternariol (AOH), alternariol-9-methyl ether (AME), altenuene (ALT) and isoaltenuene (iALT) undergo extensive oxidative metabolism, but the cytochrome P450 (CYP) isoforms responsible for the reported hydroxylation reactions are yet unknown. In the present study, the activities of twelve human CYP isoforms for the hydroxylation of AOH, AME, ALT and iALT at different positions have been determined. The most active monooxygenase for AOH and AME was CYP1A1, and lower activities were observed for CYP1A2, 2C19 and 3A4. Hydroxylation at C-2 of AOH and AME was the preferred reaction of most isoforms. For ALT and iALT, CYP2C19 had the highest activity, followed by 2C9 and 2D6. The dominating metabolite of all active isoforms was the 8-hydroxylated ALT and iALT. The activities of the CYP isoforms are consistent with the pattern of metabolites of theAlternaria toxins obtained with pooled human hepatic microsomes. Based on the activities of the CYP isoforms, a significant extrahepatic hydroxylation must be expectede.g. in the lung and esophagus for AOH and AME, and in the intestine and ovaries for ALT and iALT. As all major hydroxylation products are catechols, the extrahepatic metabolism ofAlternaria toxins may be of toxicological relevance. Presented at the 30^th Mycotoxin Workshop, Utrecht, Netheriands, April 28–30. 2008. Financial support: State of Baden-Württemberg (Research Program “Mycotoxins” as part of the Research Initiative “Food and Health”)     

Antimutagenic effects of caffeine during nitrosoguanidine-induced mutagenesis ofSalmonella typhimurium cells and phages

The effect of caffeine on nitrosoguanidine-induced mutagenesis ofSalmonella typhimurium & nd its P22 and L phages was studied. The detected mutations included phage “clear” mutations, reversions of phage “amber” mutation, and prototrophic reversions of thehis ⁻ auxotroph ofSalmonella typhimurium. Neither therecA mutation of the host nor theerf mutation of the phage genome were found to affect the nitrosoguanidine-induced mutagenesis of the phage during vegetative growth. Beginning with a concentration of 0.2 mg/ml, caffeine decreased the frequency of mutants by 30–60%, attaining a maximum effect at 1.5 mg/ml and retaining this effect even at higher concentrations. A similar antimutagenic effeot was observed with the mutagenesis of the host cells. The nitrosoguanidine-induced mutagenesis does not seem to be related to the function of therecA cell gene or theerf phage gene. The mechanism of mutagenesis by nitrosoguanidine probably has two components, one of them caffeine sensitive, the other caffeine-resistant.     

CYP21/C4 gene organisation in Italian 21-hydroxylase deficiency families

Summary A total of 33 Italian 21-hydroxylase (21-OH) deficiency families were investigated using a combination of short and long range restriction mapping of the CYP21/C4 gene cluster. The analyses revealed that large-scale length polymorphism in this gene cluster strictly conformed to a compound variable number of tandem repeats (VNTR) plus insertion system with between one and four CYP21 + C4 units and seven BssHII restriction fragment length polymorphisms (RFLPs) (75kb, 80kb, 105kb, 110kb, 135kb, 140kb and 180kb). A total of 9/66 disease haplotypes, but only 1/61 nondisease haplotypes, showed evidence of gene addition by exhibiting three or more CYP21 + C4 repeat units. Of these, two were identified in one 21-OH deficiency patient who has a total of eight CYP21 + C4 units, being homozygous for the HLA haplotype DR2 DQ2 B5 A28. This haplotype carries four CYP21 + C4 units, three of which contain CYP21A-like genes and one of which contains a CYP21B-like gene that presumably carries a pathological point mutation. Of the other gene addition haplotypes associated with 21-OH deficiency, four show three CYP21 + C4 units flanked by HLA-DR1 and HLA-B14 markers. Although such haplotypes have commonly been associated with non-classical 21-OH deficiency, three examples in the present study are unexpectedly found in two salt-wasting patients, who are respectively homozygous or heterozygous for this haplotype. Only 7/66 disease haplotypes showed evidence of a CYP21B gene deletion.     

Major histocompatibility complex (MHC) class III genetics in two Amerindian tribes from Southern Brazil: the Kaingang and the Guarani

Population genetic studies of the major histocompatibility complex (MHC) class III region, comprising C2, BF and C4 phenotypes, and molecular genetic data are rarely available for populations other than Caucasoids. We have investigated three Amerindian populations from Southern Brazil: 131 Kaingang from Ivaí (KIV), 111 Kaingang (KRC) and 100 Guarani (GRC) from Rio das Cobras. Extended MHC haplotypes were derived after standard C2, BF, C4 phenotyping and restriction fragment length polymorphism (RFLP) analysis with TaqI, together with HLA data published previously by segregation analysis. C2 and BF frequencies corresponded to other Amerindian populations. C4B*Q0 frequency was high in the GRC (0.429) but low in the Kaingang. Unusual C4 alleles were found, viz. C4A*58, A*55 and C4B*22 (presumably non-Amerindian) and aberrant C4A*3 of Amerindian origin occurring with a frequency of 0.223 in the GRC. C4A*3 bands of homo- and heterozygous individuals carrying this variant were Rodgers 1 positive and Chido 1,3 positive, showed a C4A specific lysis type and a C4A like α-chain. Polymerase chain reaction studies and sequencing showed that this is based on a C4A*3 duplication with a regular C4A*3 and a partially converted C4A*0304 carrying the C4B specific epitopes Ch 6 and Ch 1,3. Associations of class III haplotypes with particular RFLP patterns were similar to those reported for Caucasoids. The previously described association between combined C4A and CYP21P deletions and the 6.4 kb TaqI fragment was not seen in these Amerindians. This fragment occurred within a regular two locus gene structure in the Kaingang, representing a “short” gene at C4 locus I. C4 and CYP21 duplications were frequently observed. The distribution of extended MHC haplotypes provides evidence for a close relationship between the KIV and KRC and a larger genetic distance between the two Kaingang groups and the GRC. Received: 6 March 1997 / Accepted: 13 May 1997     

Molecular-genetics analysis of natural and stimulated ovulation impairment

The influence of FMR1, INHα1, NAT2, GSTT1 and GSTM1 genes mutations on ovarian function and their association with POF and “poor response” to exogenous GT after ovulation stimulation were investigated. The carriers of Ala257Thr transition predominated in the studied “poor responders” group. In 1.6% POF patients and 2.5% persons from “poor responders” group, but nobody from control group this transition combined with intermediate alleles of FMR1 gene was observed. The frequency of deletion in GSTM1 gene in “poor responders” group was significantly higher (p = 0.01) than in normal ovulatory control group. The frequency of Ser680Ser-Ala307Ala polymorphic genotype (22.2%) in “poor responders” group was significantly higher (p = 0.028) than in normal-ovulatory control group (7.7%). The daily dosage of GT in intermediate alleles of FMR1 gene carriers as well in patients with “slow acetylation” NAT2 genotype was significantly higher in comparison to patients without intermediate alleles and patients with “quick acetylation” NAT2 genotype. Quantity of oocytes after stimulation ovulation in women with INHα1 gene Ala257Thr transition were significantly decreased in comparison to patients without such mutation. Further investigations of these genes can play a major role in POF studying and modulation of ovarian response to exogenous GT. Published in Ukrainian in Tsitologiya i Genetika, 2008, Vol. 42, No. 2, pp. 63–69. The text was translated by the authors.     

The smallest value of the axon density for which ‘ignition’ can occur in a random net

As shown by A. Rapoport (1952), when a very brief stimulation or “instantaneous input” is applied to a random net, the subsequent events are determined by the parameters of the net as follows: If the axon densitya is sufficiently large and the fraction γ of the neurons initially stimulated exceeds a certain value γ₁ (theover-all threshold of the net for instantaneous stimulation), excitation will spread through the net until a steady state is reached in which a fraction γ₂ ⩾ γ₁ of the neurons is firing (“ignition phenomenon”). If γ < γ₁ the activity in the net dies out. However, if the axon density is too small, the activity will ultimately die out, no matter how large the fraction of initially stimulated neurons. Thus there exists a limiting valueA of the axon density below which the net cannot “ignite”. ThisA is a function ofh, theindividual threshold of the neurons constituting the net (we assume hereh≥2, since forh=1 the situation is essentially different). Geometrically γ₁ and γ₂ are determined as the two intersection points of a straight line with a sigmoid curve. Whena<A the two curves do not intersect and fora=A they are tangent. In this paper the “tangency case” is investigated and the general features of the functionA(h) are determined. It is shown thatA increases monotonically withh (as one would expect). For all values ofh>1 we haveA(h)>h, but the fractionA(h)/h and the derivativedA(h)/dh approach unity ash increases. An analytical expression of the functionA(h) valid for very large values ofh is derived.     

The structure ofHLA-B35 suggests that it is derived fromHLA-Bw58 by two genetic mechanisms

The primary structure ofHLA-B51 andHLA-Bw52 suggested thatHLA-B51 was derived fromHLA-Bw52 by the combination of a genetic exchange withHLA-B8 and a point mutation. To investigate the evolution of theHLA-B5 cross reactive group, theHLA-B35 gene was cloned and the primary structure was determined.HLA-B35 is identical toHLA-Bw58 except in the α₁ domain. The α₁ domain ofHLA-B35 except Bw4/Bw6-associated amino acids is identical to that ofHLA-B51 ^*, which was suspected to be an intermediate gene betweenHLA-B51 andHLA-Bw52. These data suggest thatHLA-B35 has evolved fromHLA-Bw58 in two steps; an in vivo replacement of the α₁ domain withHLA-B51 and genetic exchange with one of theHLA-Bw6 genes. These three genes andHLA-Bw58 are postulated to share a common ancestor.     

Simultaneous PCR amplification of two barley 5S rDNA sequence types in the same PCR reaction

Many researchers are currently using PCR technology to amplify individual members of multigene families, including 5S rDNA, for sequencing and related purposes. When members of the family differ in length, analyses would be facilitated if different units could be simultaneously and efficiently amplified. In the present paper we describe conditions that can be used to amplify simultaneously both the “long” and “short” 5S rDNA repeats found in barley (Hordeum vulgare L.).     

cDNA cloning and sequence analysis of the rice Cinnamate-4-Hydroxylase gene, a cytochrome P450-dependent monooxygenase involved in the general phenylpropanoid pathway

Plant cytochrome P450 monooxygenases (P450s) mediate a wide range of oxidative reactions involved in the biosynthesis of phenylpropanoids, terpenes, lipids, and alkaloids. We isolated a cDNA clone for cinnamate-4-hydroxylase (C4H) from a Japonica type rice(Oryza sativa L. cv. llpumbyeo). This C4H has a deduced amino acid sequence that is 85% identical tothat ofSorghum bicolor. Our phylogenetic analysis also showed that theOsC4HL gene is closely related toC4H fromS. bicolor. A putative genomic DNA sequence corresponding toOsC4HL contained cis-elements (boxes P, A, L, and TCA motifs), AT-rich elements, and wound-response elements that control gene expression in its promoter region.OsC4HL expression was detected in all the tissue types, with the highest level being measured in the roots. It was also apparently up-regulated by wounding stress. These data suggest that theOsC4HL gene isC4H member in theCYP73 subfamily.     

Black rat (Rattus rattus) genomic variability characterized by chromosome painting

Black rats are of outstanding interest in parasitology and infective disease analysis. We used chromosome paints from both the mouse(Mus musculus) and the Norway rat(Rattus norvegicus) to characterize the genome of two Black rat subspecies from Italy. Both subspecies have two large metacentrics (n. 1, 4) not present in the Norway rat (2n = 42).Rattus rattus rattus has a diploid number of 2n = 38, whileRattus rattus frugivorous has two small metacentric “supernumerary” or B chromosomes for a diploid number of 2n = 38 + 2B. The 21 mouse paints gave 38 signals on theR. r. rattus karyotype and 39 signals in theR. r. frugivorous karyotype. The two metacentrics, not present inR. norvegicus, were hybridized by mouse 16/1/17 and mouse 4/10/15. These chromosomes are homologous to: RRA1 = RNO 5/7, and RRA4 = RNO 9/11 and not “4/7” and “11/12” as previously reported. Furthermore, the synteny of Chr 13 of theR. r. frugivorous withR. norvegicus Chr 16 and mouse Chrs 8/14 is not complete, because there is a small pericentromeric insertion of RNO Chr 18 (mouse Chr 18). If we consider only the two metacentrics, RRA1 and RRA4, the principal differences betweenR. norvegicus andR. rattus, then we can propose the derived synteny of 124 genes in the black rat. A comparison of the Z index between rats and mice shows an acceleration of genomic evolution among genus, species, and subspecies. The chromosomal differences betweenR. r. rattus xR. r. frugivorous suggest that they may be classified as different species because hybrids would produce 50% unbalanced gametes.     

Characterization of multiple <Emphasis Type="Italic">CYP9A</Emphasis> genes in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Based on the advances in the silkworm genome project, a new genome-wide analysis of cytochrome P450 genes was performed, focusing mainly on gene duplication. All four CYP9A subfamily members from the silkworm, Bombyx mori, were cloned by RT-PCR and designated CYP9A19–CYP9A22 by the P450 Nomenclature Committee. They each contain an open reading frame of 1,593 bp in length and encode a putative polypeptide of 531 amino acids. Both nucleic acid and amino acid sequences share very high identities with one another. The typical motifs of insect cytochrome P450, including the heme-binding region, helix-C, helix-I, helix-K, and PERF, show high sequence conservation among the multiple proteins. Alignment with their cDNA sequences revealed that these paralogues share identical gene structures, each comprising ten exons and nine introns of variable sizes. The locations of their introns (all nine introns follow the GT–AG rule) are absolutely conserved. CYP9A19, CYP9A20, and CYP9A21 form a tandem cluster on chromosome 17, whereas CYP9A22 is separated from the cluster by four tandem alcohol-dehydrogenase-like genes. Their phylogenetic relationships and structural comparisons indicated that these paralogues arose as the results of gene duplication events. RT-PCR detected their mRNAs in different “first line of defense” tissues, as well as in several other organs, suggesting diverse functions. Tissue-selective expression also indicates their functional divergence. The identified CYP9A genes have not yet been found outside the Lepidoptera, and are probably unique to the Lepidoptera. They show high sequence and structural similarities to each other, indicating that the Lepidoptera-specific P450s may be of functional importance. This analysis constitutes the first report of the clustering, spatial organization, and functional divergence of P450 in the silkworm.     

MHC region and risk of systemic lupus erythematosus in African American women

The major histocompatibility complex (MHC) on chromosome 6p21 is a key contributor to the genetic basis of systemic lupus erythematosus (SLE). Although SLE affects African Americans disproportionately compared to European Americans, there has been no comprehensive analysis of the MHC region in relationship to SLE in African Americans. We conducted a screening of the MHC region for 1,536 single nucleotide polymorphisms (SNPs) and the deletion of the C4A gene in a SLE case–control study (380 cases, 765 age-matched controls) nested within the prospective Black Women’s Health Study. We also genotyped 1,509 ancestral informative markers throughout the genome to estimate European ancestry to control for population stratification due to population admixture. The most strongly associated SNP with SLE was the rs9271366 (odds ratio, OR = 1.70, p = 5.6 × 10⁻⁵) near the HLA-DRB1 gene. Conditional haplotype analysis revealed three other SNPs, rs204890 (OR = 1.86, p = 1.2 × 10⁻⁴), rs2071349 (OR = 1.53, p = 1.0 × 10⁻³), and rs2844580 (OR = 1.43, p = 1.3 × 10⁻³), to be associated with SLE independent of the rs9271366 SNP. In univariate analysis, the OR for the C4A deletion was 1.38, p = 0.075, but after simultaneous adjustment for the other four SNPs the odds ratio was 1.01, p = 0.98. A genotype score combining the four newly identified SNPs showed an additive risk according to the number of high-risk alleles (OR = 1.67 per high-risk allele, p < 0.0001). Our strongest signal, the rs9271366 SNP, was also associated with higher risk of SLE in a previous Chinese genome-wide association study (GWAS). In addition, two SNPs found in a GWAS of European ancestry women were confirmed in our study, indicating that African Americans share some genetic risk factors for SLE with European and Chinese subjects. In summary, we found four independent signals in the MHC region associated with risk of SLE in African American women.     

Ecological Segregation and Population Structuring of the Cormorant Phalacrocorax carbo in Europe, in Relation to the Recent Introgression of Continental and Marine Subspecies

Populations of the “continental” Great Cormorant P. c. sinensis have expanded from north-eastern Europe towards the western part of the range of the “marine” P. c. carbo breeding in the United Kingdom and France. The aim of the study was to test the hypothesis of ecological segregation between subspecies by analysing the structuring of the European populations. Sequencing the mtDNA of 231 birds belonging to 20 colonies revealed 38 haplotypes based on 25 polymorphic sites (5.76% sequence divergence). P. c. sinensis (“S”) was well confirmed, but usual P. c. carbo formed two coastal populations, the real P. c. carbo “C” mainly in the western part of the range (United Kingdom, coastal France), and also in Norway and Sardinia, and “N”, branched to the Japanese Cormorant P. capillatus and probably isolated by glaciations, mainly present in the Nordic range (Norway, but also on the coasts from Sweden to Brittany), we named P. c. norvegicus. In a variable position in the trees but close to C is a group of undetermined origin haplotypes, named U, also present in both traditional ranges. The new tree-nesting colonies in Brittany are clearly a mixture of S and the two clades C and N previously described as P. c. carbo, with a decreasing proportion of C + N between 1993 (67%), 1996 (60%) and 2002 (33%) for the pioneering Grand-Lieu colony. These results confirmed the current introgression of continental populations in the western range, with probable hybridization. Although the subspecies can switch habitats locally due to social behaviour and migrations, the ecological segregation between the two usual subspecies appears to be largely confirmed in Europe.     

Order of class III genes relative to HLA genes determined by the haplotype method

The B18 C4A3 C4BQ0 BfF1 DR3 haplotype was found to be ideal for determining the order of C4 and Bf relative to HLA-B and DR by the haplotype method. All the copies of this haplotype are assumed to be derived from a single ancestral haplotype. Sixteen of the twenty-six BfFl-containing haplotypes carried all of the alleles from this ancestral haplotype. Most of the other BfFl-containing haplotypes could be derived from the ancestral haplotype by a single crossover event for one of the two possible gene orders. This suggests that B18 C4A3 C4BQ0 BfFl DR3 is the sole source of the BfFl allele. The uncommon C4 type on B18 C4A3 C4BQ0 BfFl DR3 facilitates recognition of the BfFl-containing products of recombination between Bf and C4. One such recombinant haplotype was found which shows that the orientation of the class III genes is as follows: C4 is closest to HLA-B and Bf is closest to HLA-DR. This gene order is supported by all the earlier unequivocal results obtained using the haplotype method (Olaisen et al. 1983, Marshall et al. 1984a). Combining these results with the information on class III genes obtained from overlapping cosmid clones (Carroll et al. 1984) and earlier mapping studies (Robson and Lamm 1984) shows that HLA-B is telomeric to 21B. C4B, 21A, C4A, Bf and C2 then follow 21B in that order covering 120 kb. HLA-DR is located further toward the centromere.     

Detection of gene flow from GM to non-GM watermelon in a field trial

Gene flow from genetically modified (GM) crops to conventional non-GM crops is a serious concern for protection of conventional and organic farming. Gene flow from GM watermelon developed for rootstock use, containing cucumber green mottle mosaic virus (CGMMV)-coat protein (CP) gene, to a non-GM isogenic control variety “Clhalteok” and grafted watermelon “Keumcheon” was investigated in a small scale field trial as a pilot study. Hybrids between GM and non-GM watermelons were screened from 1304 “Chalteok” seeds and 856 “Keumcheon” seeds using the duplex PCR method targeting theCGMMV- CP gene as a marker. Hybrids were found in all pollen recipient plots. The gene flow frequencies were greater for “Chaiteok” than for “KeumcheonD; with 75% outcrossing in the “Chaiteok” plot at the closest distance (0.8 m) to the GM plot. A much larger scale field trial is necessary to identify the isolation distance between GM and non-GM watermelon, as the behaviors of insect pollinators needs to be clarified in Korea.