Pulsed-field Gel Electrophoresis Experiments Research Articles

Miniaturized System for Rapid Field Inversion Gel Electrophoresis of DNA with Real-Time Whole-Gel Detection

Pulsed field gel electrophoresis (PFGE) methods have become standard tools in a wide range of DNA analysis applications. But many aspects of DNA migration phenomena under pulsed field conditions are not well understood as compared with the more conventional situation where the electric field is held constant. A key reason for this deficiency is that PFGE experiments are cumbersome to perform due to extremely long separation times (approximately 10-15 h) and the need to perform gel analysis by poststaining after completion of the run. Here we introduce an easy to build miniaturized slab gel apparatus that addresses these issues by enabling large DNA fragments up to 35 kb in length to be separated using field inversion gel electrophoresis (FIGE) in 60-90 min. The compact size of the device also allows the entire gel to be continuously monitored so that the separation processes can be imaged in real time using a high-resolution CCD camera. Arbitrary control over the applied voltage waveforms is achieved using a function generator interfaced with a high voltage amplifier. These capabilities allow us to probe the size dependence of fundamental physical parameters associated with DNA migration (mobility, diffusion, and separation resolution). These data reveal a surprising regime where separation resolution increases with DNA fragment size owing to a favorable interplay between mobility and diffusion scalings and highlight the important role of diffusion (a seldom quantified parameter). In addition to the practical benefit of separation times that are an order of magnitude faster than conventional instruments, the results of these studies provide a previously unavailable rational basis to identify optimal separation conditions and contribute new insights toward understanding the underlying physical processes that govern DNA electrophoresis in pulsed fields.

The Genome of Nectria haematococca: Contribution of Supernumerary Chromosomes to Gene Expansion

The ascomycetous fungus Nectria haematococca, (asexual name Fusarium solani), is a member of a group of >50 species known as the “Fusarium solani species complex”. Members of this complex have diverse biological properties including the ability to cause disease on >100 genera of plants and opportunistic infections in humans. The current research analyzed the most extensively studied member of this complex, N. haematococca mating population VI (MPVI). Several genes controlling the ability of individual isolates of this species to colonize specific habitats are located on supernumerary chromosomes. Optical mapping revealed that the sequenced isolate has 17 chromosomes ranging from 530 kb to 6.52 Mb and that the physical size of the genome, 54.43 Mb, and the number of predicted genes, 15,707, are among the largest reported for ascomycetes. Two classes of genes have contributed to gene expansion: specific genes that are not found in other fungi including its closest sequenced relative, Fusarium graminearum; and genes that commonly occur as single copies in other fungi but are present as multiple copies in N. haematococca MPVI. Some of these additional genes appear to have resulted from gene duplication events, while others may have been acquired through horizontal gene transfer. The supernumerary nature of three chromosomes, 14, 15, and 17, was confirmed by their absence in pulsed field gel electrophoresis experiments of some isolates and by demonstrating that these isolates lacked chromosome-specific sequences found on the ends of these chromosomes. These supernumerary chromosomes contain more repeat sequences, are enriched in unique and duplicated genes, and have a lower G+C content in comparison to the other chromosomes. Although the origin(s) of the extra genes and the supernumerary chromosomes is not known, the gene expansion and its large genome size are consistent with this species' diverse range of habitats. Furthermore, the presence of unique genes on supernumerary chromosomes might account for individual isolates having different environmental niches.

Comparison ofStreptococcus mutansstrains from children with caries-active, caries-free and gingivitis clinical diagnosis by pulsed-field gel electrophoresis

A study was conducted to compare the DNA structure of Streptococcus mutans strains in children with caries-active, caries-free, and gingivitis clinical diagnosis. Twenty-eight Streptococcus mutans strains from 100 children's plaques were examined by pulsed-field gel electrophoresis (PFGE) method. The classified strains were closely related to one another, though the strains originated from different disease groups. Three identical pairs were found, but the pairs in two cases belonged to different disease groups. The results of the PFGE experiments suggest that there is no correlation between the different DNA patterns ofS. mutans strains and their cariogenecity. So the different DNA strains ofS. mutans are not the only determining factor in the development of dental caries.

Subtraction of background damage in PFGE experiments on DNA fragment-size distributions

The non-random distribution of DNA breakage in pulsed-field gel electrophoresis (PFGE) experiments poses a problem of proper subtraction of the background damage to obtain a fragment-size distribution due to radiation only. As been pointed out by various authors, a naive bin-to-bin subtraction of the background signal will not result in the right DNA mass distribution histogram, and may even result in negative values. Previous more systematic subtraction methods have been based mainly on random breakage, appropriate for low-LET radiation but problematic for high LET. Moreover, an investigation is needed whether the background breakage itself is random or non-random. Previously a new generalized formalism based on stochastic processes for the subtraction of the background damage in PFGE experiments for any LET and any background was proposed, and as now applied it to a set of PFGE data for Fe ions. We developed a Monte Carlo algorithm to compare the naïve subtraction procedure in artificial data sets to the result produced by the new formalism. The simulated data corresponded to various cases, involving non-random (high-LET) or random radiation breakage and random or non-random background breakage. The formalism systematically gives better results than naïve bin-by-bin subtraction in all these artificial data sets.

A Robust Procedure for Removing Background Damage in Assays of Radiation-Induced DNA Fragment Distributions

The non-random distribution of DNA breakage in PFGE (pulsed-field gel electrophoresis) experiments poses a problem of proper subtraction of the background DNA damage to obtain a fragment-size distribution due to radiation only. A naive bin-to-bin subtraction of the background signal will not result in the right DNA mass distribution histogram. This problem could become more pronounced for high-LET (linear energy transfer) radiation, because the fragment-size distribution manifests a higher frequency of smaller fragments. Previous systematic subtraction methods have been based on random breakage, appropriate for low-LET radiation. Moreover, an investigation is needed to determine whether the background breakage is itself random or non-random. We consider two limiting cases: (1) the background damage is present in all cells, and (2) it is present in only a small subset of cells, while other cells are not contributing to the background DNA fragmentation. We give a generalized formalism based on stochastic processes for the subtraction of the background damage in PFGE experiments for any LET and apply it to two sets of PFGE data for iron ions.

Association of beta2 toxin production with Clostridium perfringens type A human gastrointestinal disease isolates carrying a plasmid enterotoxin gene

Clostridium perfringens type A isolates carrying an enterotoxin (cpe) gene are an important cause of human gastrointestinal diseases, including food poisoning, antibiotic-associated diarrhoea (AAD) and sporadic diarrhoea (SD). Using polymerase chain reaction (PCR), the current study determined that the cpb2 gene encoding the recently discovered beta2 toxin is present in <15% of food poisoning isolates, which typically carry a chromosomal cpe gene. However, >75% of AAD/SD isolates, which usually carry a plasmid cpe gene, tested cpb2(+) by PCR. Western blot analysis demonstrated that >97% of those cpb2(+)/cpe(+) AAD/SD isolates can produce CPB2. Additional PCR analyses, sequencing studies and pulsed field gel electrophoresis experiments determined that AAD/SD isolates carry cpb2 and cpe on the same plasmid when IS1151 sequences are present downstream of cpe, but cpb2 and cpe are located on different plasmids in AAD/SD isolates where IS1470-like sequences are present downstream of cpe. Those analyses also demonstrated that two different CPB2 variants (named CPB2h1 or CPB2h2) can be produced by AAD/SD isolates, dependent on whether IS1470-like or IS1151 sequences are present downstream of their cpe gene. CPB2h1 is approximately 10-fold more cytotoxic for CaCo-2 cells than is CPB2h2. Collectively, these results suggest that CPB2 could be an accessory toxin in C. perfringens enterotoxin (CPE)-associated AAD/SD.

A new Bacteroides conjugative transposon that carries an ermB gene.

The erythromycin resistance gene ermB has been found in a variety of gram-positive bacteria. This gene has also been found in Bacteroides species but only in six recently isolated strains; thus, the gene seems to have entered this genus only recently. One of the six Bacteroides ermB-containing isolates, WH207, could transfer ermB to Bacteroides thetaiotaomicron strain BT4001 by conjugation. WH207 was identified as a Bacteroides uniformis strain based on the sequence of its 16S rRNA gene. Results of pulsed-field gel electrophoresis experiments demonstrated that the transferring element was normally integrated into the Bacteroides chromosome. The element was estimated from pulsed-field gel data to be about 100 kb in size. Since the element appeared to be a conjugative transposon (CTn), it was designated CTnBST. CTnBST was able to mobilize coresident plasmids and the circular form of the mobilizable transposon NBU1 to Bacteroides and Escherichia coli recipients. A 13-kb segment that contained ermB was cloned and sequenced. Most of the open reading frames in this region had little similarity at the amino acid sequence level to any proteins in the sequence databases, but a 1,723-bp DNA segment that included a 950-bp segment downstream of ermB had a DNA sequence that was virtually identical to that of a segment of DNA found previously in a Clostridium perfringens strain. This finding, together with the finding that ermB is located on a CTn, supports the hypothesis that CTnBST could have entered Bacteroides from some other genus, possibly from gram-positive bacteria. Moreover, this finding supports the hypothesis that many transmissible antibiotic resistance genes in Bacteroides are carried on CTns.

Presence of the tet (O) Gene in Erythromycin- and Tetracycline-Resistant Strains of Streptococcus pyogenes and Linkage with either the mef (A) or the erm (A) Gene

Sixty-three recent Italian clinical isolates of Streptococcus pyogenes resistant to both erythromycin (MICs >or=1 microg/ml) and tetracycline (MICs >or= 8 microg/ml) were genotyped for macrolide and tetracycline resistance genes. We found 19 isolates carrying the mef(A) and the tet(O) genes; 25 isolates carrying the erm(A) and tet(O) genes; and 2 isolates carrying the erm(A), tet(M), and tet(O) genes. The resistance of all erm(A)-containing isolates was inducible, but the isolates could be divided into two groups on the basis of erythromycin MICs of either >128 or 1 to 4 microg/ml. The remaining 17 isolates included 15 isolates carrying the erm(B) gene and 2 isolates carrying both the erm(B) and the mef(A) genes, with all 17 carrying the tet(M) gene. Of these, 12 carried Tn916-Tn1545-like conjugative transposons. Conjugal transfer experiments demonstrated that the tet(O) gene moved with and without the erm(A) gene and with the mef(A) gene. These studies, together with the results of pulsed-field gel electrophoresis experiments and hybridization assays with DNA probes specific for the tet(O), erm(A), and mef(A) genes, suggested a linkage of tet(O) with either erm(A) or mef(A) in erythromycin- and tetracycline-resistant S. pyogenes isolates. By amplification and sequencing experiments, we detected the tet(O) gene ca. 5.5 kb upstream from the mef(A) gene. This is the first report demonstrating the presence of the tet(O) gene in S. pyogenes and showing that it may be linked with another gene and can be moved by conjugation from one chromosome to another.

A newly discovered Bacteroides conjugative transposon, CTnGERM1, contains genes also found in gram-positive bacteria.

Results of a recent study of antibiotic resistance genes in human colonic Bacteroides strains suggested that gene transfer events between members of this genus are fairly common. The identification of Bacteroides isolates that carried an erythromycin resistance gene, ermG, whose DNA sequence was 99% identical to that of an ermG gene found previously only in gram-positive bacteria raised the further possibility that conjugal elements were moving into Bacteroides species from other genera. Six of seven ermG-containing Bacteroides strains tested were able to transfer ermG by conjugation. One of these strains was chosen for further investigation. Results of pulsed-field gel electrophoresis experiments showed that the conjugal element carrying ermG in this strain is an integrated element about 75 kb in size. Thus, the element appears to be a conjugative transposon (CTn) and was designated CTnGERM1. CTnGERM1 proved to be unrelated to the predominant type of CTn found in Bacteroides isolates-CTns of the CTnERL/CTnDOT family-which sometimes carry another type of erm gene, ermF. A 19-kbp segment of DNA from CTnGERM1 was cloned and sequenced. A 10-kbp portion of this segment hybridized not only to DNA from all the ermG-containing strains but also to DNA from strains that did not carry ermG. Thus, CTnGERM1 seems to be part of a family of CTns, some of which have acquired ermG. The percentage of G+C content of the ermG region was significantly lower than that of the chromosome of Bacteroides species-an indication that CTnGERM1 may have entered Bacteroides strains from some other bacterial genus. A survey of strains isolated before 1970 and after 1990 suggests that the CTnGERM1 type of CTn entered Bacteroides species relatively recently. One of the genes located upstream of ermG encoded a protein that had 85% amino acid sequence identity with a macrolide efflux pump, MefA, from Streptococcus pyogenes. Our having found >90% sequence identity of two upstream genes, including mefA, and the remnants of two transposon-carried genes downstream of ermG with genes found previously only in gram-positive bacteria raises the possibility that gram-positive bacteria could have been the origin of CTnGERM1.

Phenotypic and genetic characterisation of bacteriocin-producing strains of Staphylococcus aureus involved in bovine mastitis

Fifty strains of Staphylococcus spp. isolated from bovine mastitis cases in several herds from different Argentinian provinces were screened for antimicrobial substances. Twelve strains exhibited a high antagonistic activity against the indicator strain ( Corynebacterium fimi) and were chosen for further characterisation. The antimicrobial substances were sensitive to proteolytic enzymes suggesting that they might be bacteriocins (Bac). These strains were identified as S. aureus by the amplification of the femA gene. Plasmid profile analysis of these strains revealed the presence of at least one plasmid. Eleven strains carried a plasmid with a size similar to that of pRJ6 (8.0 kb), which encodes aureocin A70, a bacteriocin produced by the Brazilian S. aureus strain A70 isolated from commercial milk. The other strain harboured a much larger plasmid. PCR experiments, using specific primers for amplification of the bacteriocin operon found in pRJ6, showed that all strains had the expected 525 bp amplicon, suggesting that the bacteriocin produced may be related to aureocin A70. The genomic DNA of all Bac + strains was then analysed by pulsed-field gel electrophoresis (PFGE) in order to investigate clonal relationships amongst strains. Based on the results of PFGE experiments, 10 out of the 12 Bac + strains belonged to the same clone. The remaining two strains are possibly related to the prevalent clone. The aureocin A70 producer-strain belonged to a distinct clone.

Outbreak of infection with extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a Mexican hospital.

Thirty-one strains of Klebsiella pneumoniae (including 10 duplicates) from 21 septicemic pediatric patients (age, <2 months) were studied during a 4-month period (June to October 1996) in which the fatality rate was 62% (13 of 21). These isolates identified by the API 20E system yielded the same biotype. Pulsed-field gel electrophoresis experiments revealed the same clone in 31 strains. The isolates were multidrug-resistant but were still susceptible to ciprofloxacin, imipenem, and cefoxitin. A 135-kb plasmid was harbored in all of the isolates. No transconjugants were obtained that were resistant to ampicillin, cefotaxime, tetracycline, or gentamicin. Isoelectric focusing for beta-lactamases was performed on all strains, and three bands with pIs of 5.4, 7.6, and 8.2 were obtained. Of these, the pI 8.2 beta-lactamase had an extended-spectrum beta-lactamase phenotype. PCR amplification of both TEM- and SHV-type genes was obtained. The sequence analysis of the SHV PCR product indicated a mutation corresponding to the SHV-5 beta-lactamase.

GENETIC ANALYSIS OF THE BLACKLEG FUNGUS LEPTOSPHAERIA MACULANS

A genetic and physical map has been developed for Leptosphaeria maculans, which causes blackleg of oilseed Brassicas. The genetic map was constructed from 58 F1 progeny and comprises 154 Amplified Fragment Length Polymorphic (AFLP) markers, 3 Random Amplified Polymorphic DNA (RAPD) markers, a gene encoding a retinal-binding protein, the mating type locus and a host specificity locus. Pulsed field gel electrophoresis experiments showed that the parental isolates each had 16 chromosomes and a genome size of about 33.5 Mb. Many markers bound to every chromosome indicating that L. maculans has a high level of dispersed repetitive DNA sequences. This fungus displays chromosomal length polymorphisms, but in the cross examined, the linkage and physical maps were congruent and there was no evidence of translocations. The host specificity locus is flanked by two AFLP markers 19 and 27 cM distant and is located on a chromosome sized 1.85 Mb in the virulent parent. The mating type locus is on a chromosome sized 2.6 Mb and co-incident on an AFLP marker amplified from the virulent parent.

Genome analysis of the plant pathogenic ascomycete Leptosphaeria maculans; mapping mating type and host specificity loci

Abstract A genetic and physical map has been developed for the loculoascomycete Leptosphaeria maculans, a pathogen of oilseed Brassicas. The genetic map was constructed from 58 F(1) progeny and comprises 155 amplified fragment length polymorphic (AFLP) markers, three random amplified polymorphic DNA (RAPD) markers, the mating type locus and a host specificity locus conferring the ability to form lesions on Brassica juncea. Twenty-one linkage groups, 5 pairs, and 18 unlinked markers were assigned, and the genome size was 1520 cM. Pulsed field gel electrophoresis experiments showed that the parental isolates each had 16 chromosomes and a genome size of about 33.5 Mb. Attempts to anchor a large number of markers to chromosomes were hampered by difficulties in converting AFLPs into RFLP markers, and because many markers bound to every chromosome, indicating that L. maculans has a high level of dispersed repetitive sequences. This fungus displays chromosomal length polymorphisms, but in the cross examined, the linkage and physical maps were essentially congruent and there was no evidence of translocations. The host specificity locus is 18 cM from the nearest AFLP marker and is located on a chromosome sized 1.85 Mb in the virulent parent. The mating type locus is on a chromosome sized 2.6 Mb and coincident on an AFLP marker amplified from the virulent parent. The derived amino acid sequence of part of this marker has some conserved amino acids present in the High Mobility Group DNA binding domain of MAT-2 mating type genes of other ascomycetes.

Quantification of DNA damage by PFGE: development of an analytical approach to correct for the background distribution

Purpose : To analyse the currently existing methods to infer the extent of cellular DNA damage induced by ionizing radiation when the pulsed field gel electrophoresis (PFGE) technique is used. Results and conclusions : PFGE is currently the method of choice for the measurement of radiation-induced double-strand breaks (dsb). For accurate determination of both the yields and distributions of breaks, separation of a large range of fragment sizes is required. In the conventional analysis of PFGE experiments, the background distribution of fractionated molecules is, normally, simply subtracted from the irradiated measured distribution, for each molecular weight region available. This work shows that this approach may lead to incorrect estimation of the breakage frequencies. An alternative approach based on correcting the fitting functions for the actual nonrandom damage present in the control unirradiated samples has been developed.

A Physical, Transcript, and Deletion Map of Chromosome Region 12p12.3 Flanked byETV6andCDKN1B:Hypermethylation of theLRP6CpG Island in Two Leukemia Patients with Hemizygous del(12p)

FISH analyses and loss of heterozygosity studies have delineated a commonly deleted region in hematological malignancies flanked byETV6andCDKN1Bon chromosome 12p12.3. The same chromosomal region is also a target for deletions in certain solid tumors. As an initial step toward the cloning of a potential tumor suppressor gene at 12p12.3, we mapped theETV6–CDKN1Bregion physically using bacterial artificial chromosome (BAC) and P1-derived clone (PAC) contigs. The 1.2-Mb high-resolution, contiguous map extends from D12S1095 to D12S929 and consists of 19 PACs and 20 BACs. Pulsed-field gel electrophoresis experiments confirmed the integrity of the clone-based map and identified six CpG islands in the region. A transcript map was generated by performing hybridization selection experiments with the genomic clones, by evaluating known 12p ESTs for their presence in the contig, and by sequence analysis of CpG islands in the region. Altogether evidence was gathered for the presence of the recently publishedLRP6gene and at least seven other new genes in this chromosomal region. TheCLAPS3gene, mapped between D12S391 and D12S358, was reassigned to chromosome 5 since genomic sequencing demonstrated the chromosome 12p sequence to be a pseudogene. Polymorphic CA repeats were identified approximately every 100 kb, which will support future analysis of loss of heterozygosity in tumors. Fluorescencein situhybridization analysis of leukemia patients with del(12p) further refined the commonly deleted segment to 600 kb betweenETV6and D12S358, which apparently excludesCDKN1B.Methylation changes of the CpG islands in theETV6–CDKN1Binterval were assessed by Southern analysis for leukemia patients with hemizygous 12p deletions. A “de novo” methylation was detected only at theLRP6CpG island in 2 of 22 leukemia patients tested and was confirmed by methylation-sensitive PCR and sequencing. The genomic structure ofLRP6was elucidated to allow screening for inactivating mutations, but only intragenic polymorphisms were identified. Hypermethylation of CpG islands associated with gene promoters is reported as a common mechanism for gene silencing and tumor suppressor inactivation. Therefore the consequences of theLRP6CpG island methylation and its role in the observed phenotype need further investigation.

A Plasmodium falciparum homologue of the ATPase subunit of a multi-protein complex involved in chromatin remodelling for transcription

A Plasmodium falciparum homologue of one of the components of a chromatin-remodelling complex which controls binding of transcription factors to nucleosome core particles has been cloned and characterised. The gene encodes 1422 amino acids with an estimated molecular mass of 167 kDa. The protein, SNF2L, shares 60% amino acid identity in its conserved DNA-dependent ATPase domain with yeast transcription factors originally identified by characterising mating type switch mutants. It also contains sequences related to the so-called SWI3, ADA2, N-CoR and TFIIIB B″ or SANT DNA binding domains which are characteristic of these transcriptional activation factors. The SNF2L gene has two short introns in the 3′ region of the coding sequence of the gene and is transcribed into a single ∼6.5 kb messenger RNA species which is present throughout the asexual stages of the cell cycle. Southern blotting and pulsed field gel electrophoresis experiments show that SNF2L is a single copy gene, located on P. falciparum chromosome 11.

Real-time imaging of the reorientation mechanisms of YOYO-labelled DNA molecules during 90 degrees and 120 degrees pulsed field gel electrophoresis.

Pulsed field gel electrophoresis (PFGE) techniques have been developed to overcome the limitations of conventional electrophoresis and to increase the separation to DNA chromosomes of few megabase pairs in size. Despite of the large success of these techniques, the various separation protocols employed for PFGE experiments have been determined empirically. However, a deep understanding of the molecular mechanisms of motion responsible for DNA separation becomes necessary for the rational optimization of these techniques. This paper shows the first clear observations of individual molecules of DNA during the reorientation process in 90 degrees PFGE and 120 degrees PFGE. Real-time visualization of the DNA dynamics during PFGE was possible with the use of an epi-illumination fluorescence microscope specifically equipped to run these experiments and by staining the DNA with YOYO-1 (1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-meth yl -2,3-dihydro-(benzo-1,3-oxazole)-2-methyl-idene]-quinolinium tetraiodide). This dye forms a very stable, highly fluorescent complex with double-stranded DNA and dramatically improves the quality of the DNA images. The results of computer simulations used to reproduce the molecular mechanisms of motion as well as the DNA separation features are also discussed.

Inheritance of chromosomal length polymorphisms in the ascomycete Leptosphaeria maculans.

Pulsed field gel electrophoresis experiments show that chromosomal length polymorphisms are produced during meiosis in the ascomycetous plant pathogen Leptosphaeria maculans. Homologues in tetrads of L. maculans were identified on the basis of their binding to chromosome-specific probes that included beta-tubulin, nitrate reductase, 18S ribosomal DNA and two Random Amplified Polymorphic DNA (RAPD) markers. Changes in size of homologues were followed during meiosis. Significant karyotype variation was evident due to the random assortment of parental homologues of different sizes. In most cases, the progeny had the same-sized homologues as the parents; however, in some instances novel-sized homologues were detected that varied in size from those of the parents by up to 50 kb. Our results are consistent with the hypothesis that these novel chromosomal length polymorphisms are produced by reciprocal recombination between parental homologous chromosomes of unequal sizes.

The immunoglobulin κ locus of primates

The immunoglobulin κ genes of nonhuman primates were studied by using sequence information and hybridization probes derived from the human κ gene regions. The following results were obtained: (1) V κ gene probes of the three major human κ subgroups hybridized to restriction nuclease digests of DNA from the chimpanzees Pan troglodytes (PTR) and Pan paniscus (PPA), the gorilla Gorilla gorilla (GGO), the orangutan Pongo pygmaeus (PPY), the macaque Macaca mulatta (MMU), the marmoset Callithrix geoffrei (CGE), and the bushbaby Galago demidovii (GDE), yielding patterns of decreasing similarity to the patterns of the human V κ multigene family. (2) The C κ gene segments of PTR, GGO, and PPY were 99.6, 97, and 93%, respectively, identical in sequence to the human C κ gene. A V κ gene in PTR, GGO, PPY, and MMU was 98, 96, 96, and 95%, respectively, identical to the most C κ proximal V κ gene, called B3. The other two J κC κ proximal V κ genes in human, B1 and B2, hybridize to restriction fragments of sizes identical to that of DNA from humans and great apes. (3) The long-range restriction maps of the human (HSA), PTR, and GGO κ loci as established by pulsed-field gel electrophoresis (PFGE) are quite homologous. According to the maps, however, and to hybridization studies with 11 duplication-differentiating probes, there is only one copy of the locus in PTR and GGO. This means that the duplication of large parts of the κ locus as found in humans occurred after the branch-point of human and great ape evolution. Despite the high similarity in structural details, the κ locus of the chimpanzee probably comprises only about half as many V κ genes as the human κ locus. (4) Comparative PFGE experiments indicate that a V κ-orphon region on the long arm of chromosome 2 (cos108) was translocated by a pericentric inversion that occurred after the separation of the GGO and PPY from the HSA and PTR clades.

The Met/Hepatocyte growth factor receptor (HGFR) gene is overexpressed in some cases of human leukemia and lymphoma

The proto-oncogene c- met encodes a heterodimeric (α, β) tyrosine kinase receptor which binds the hepatocyte growth factor (HGF). Recently, overexpression of the Met/HGF receptor gene has been detected in fresh samples of carcinomas and in epithelial tumor cell lines but not in cell lines derived from human leukemia and lymphoma. Our analysis of 50 primary samples of human leukemia and lymphoma and 23 hematopoietic cell lines revealed expression of mRNA and protein of the met/HGF receptor in 6 out of the 73 hematopoietic tumor samples analyzed. Four of the six samples positive for expression of the Met/HGF receptor gene were derived from patients with Hodgkin's disease. In addition, in one Burkitt's lymphoma cell line and in one acute myeloid leukemia (AML), expression of the Met/HGF receptor gene was detected. In normal unstimulated lymphocytes, granulocytes or monocytes we did not find expression of the Met/HGF receptor gene. Upon stimulation with the phorbol ester TPA we detected a weak expression of Met/HGF receptor specific transcripts of 9.0 kb in peripheral blood mononuclear cells of a healthy donor. Cytogenetic analyses of three of the four cell lines which express the Met/HGF receptor gene revealed structural or numerical abnormalities of the long arm of chromosome 7, where the Met/HGFR gene is located, in each of the three cell lines analyzed. In one of these cell lines (L540) the Met/HGFR gene is translocated to a marker chromosome. Southern blot and pulsed field gel electrophoresis experiments did not show any rearrangement in a region of 600 kb around the Met/HGF receptor gene excluding an activation of Met/HGFR by a TPR/Met oncogenic rearrangement as described for MNNG-HOS cells and for some gastric tumors. Our data indicate that the Met/HGFR gene is deregulated in a few cases of human leukemia, Burkitt's lymphoma and Hodgkin's disease possibly by chromosomal rearrangements resulting in an overexpression of the normal Met/HGF receptor mRNA and protein without formation of a hybrid gene.