Association of polymorphisms in the HLA-B region with extended haplotypes

Genomic probes from the HLA-B region of the major histocompatibility complex (MHC) were used to study the association of restriction fragment length polymorphisms (RFLPs) with various MHC alleles, complotypes, and extended haplotypes. The two DNA probes, M20A and R5A, were derived from previously cloned cosmids and are located 38 and 110 kilobases (kb) centromeric to HLa-B, respectively. Five different RFLP variants occuring in five different haplotypic combinations were detected within a panel of 40 homozygous-typing cells and cells from 21 families using Bst EII. In two informative families with HLA-B/DR recombinations the inheritance of the RFLP variants was consistent with their mapping between HLA-B and complotypes. The R5A/M20A haplotypic pattern 6.5 kb/3.0 kb (A) had a normal Caucasian frequency of approximately 0.43 and was found in all independent examples of the extended haplotypes [HLA-B8,SC01,DR3], [HLA-B18,F1C30, DR3], [HLa-Bw62,SC33,Dr4], [HLa-B44,SC30,Dr4], and [HLA-Bw47,FC91,0DR7]. The patterns of 6.9 kb/ 3.0 kb (B), 6.5 kb/4.7 kb (C), 1.45 kb/3.0 kb (D), and 6.9 kb/4.7 kb (E) had normal Caucasian frequencies of approximately 0.23, 0.15, 0.15, and 0.04 and were found on all independent examples of [HLA-B38,SC21, DR4], [HLA-Bw57,SC61,DR7], [HLA-B7,SC31,DR2], and [HLA-B44,FC31,DR7], respectively. Individual complotypes or HLA-B alleles which were markers of extended haplotypes showed variable associations. For example, HLA-B7 and the complotype SC31 were associated with all R5A/M20A RFLP haplotypes except haplotype E, although [HLA-B7,SC31,DR7] was associated exclusively with haplotype D. HLA-B27, not known to be part of an extended haplotype, was suprisingly exclusively associated with the 6.5 kb/4.7 kb or C haplotypic pattern in all five instances tested. These findings support the concept of regional conservation of DNA on independent examples of extended haplotypes. The results also further characterize these haplotypes.     

Nuclear DNA polymorphisms in a wild population of yellow baboons (Papio hamadryas cynocephalus) from Mikumi National Park,Tanzania

Yellow baboons (Papio hamadryas cynocephalus) from Mikumi National Park, Tanzania were studied for polymorphisms in nuclear DNA. The study population consists of four social groups that inhabit overlapping home ranges and exchange males. As a result, these groups are considered to be members of a single interbreeding population. Human DNA clones were used as probes to screen five loci (AT3, REN, HEXB, VIM, and APOB) for restriction fragment length polymorphisms (RFLP). A total of 14 polymorphisms, at least one at each locus, was detected in a panel of 27 baboons tested using six restriction enzymes for each locus. Eleven of these RFLP systems have average heterozygosity values greater than 0.40. This initial screening demonstrates that human DNA clones can be used to detect significant numbers of informative DNA polymorphisms in single-copy nuclear genes of this species and suggests that the average proportion of nucleotides polymorphic across nuclear loci in this population may be between 1.0% and 1.3%. © 1993 Wiley-Liss, Inc.     

Restriction fragment length polymorphism of the human T cell receptor alpha gene

Previous studies have demonstrated restriction fragment length polymorphisms (RFLP) in the vicinity of the alpha and beta genes of the human T-cell receptor. In the course of experiments designed to discover additional polymorphic restriction sites, we found a new RFLP of the T-cell alpha gene recognized by the restriction enzyme Taq I. The site was localized to the interval between the most 3 joining (J) exon and the most 5 constant (C) region exon, about 7 kb distant from the previously described Bgl II polymorphic site which mapped to the vicinity of the 3 untranslated exon. With the use of these two polymorphic markers, four Ti-alpha alleles could be identified, allowing unambiguous assignment of all Ti-alpha genes in some families. These markers may be useful in identifying possible immune response genes or disease predisposition genes associated with the genes of the T-cell receptor for antigen.Abbreviations used in this paper RFLP restriction fragment length polymorphism - Ti-alpha alpha gene of the T-cell receptor for antigen     

HLA-DR genotyping by restriction fragment length polymorphism analyses

We have established unique restriction fragment length polymorphism (RFLP) patterns characteristic of homozygous typing cells (HTCs) for HLA-DR-1 through HLA-DR-8 haplotypes. These RFLP patterns were found to segregate in family members and correlate 100% with HLA-DR antibody phenotyping. The RFLP patterns were used to type chronic myelocytic leukemic cells which have a Philadelphia translocation from 23 randomly selected Caucasoid patients. The results show an alternative method for the determination of the HLA-DR types without using live cells and to study disease association with the HLA-DR region.     

Restriction fragment length polymorphism of the major histocompatibility complex of the dog

Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D-homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (Bam HI, Eco RI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I, and Bgl II), separated by agarose gel electrophoresis, and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabeled HLA cDNA probes corresponding to DR, DQ, DP, and DO beta genes. The autoradiograms for all nine enzyme digests displayed multiple bands with the DRb, DQb, and DPb probes while the DOb probe hybridized with one to two bands. The RFLP patterns were highly polymorphic but consistent within each DLA-D type. Standard RFLP patterns were established for nine DLA-D types which could be discriminated from each other by using two enzymes (Rsa I and Pst I) and the HLA-DPb probe. Cluster analysis of the polymorphic restriction fragments detected by the DRb probe revealed four closely related supertypic groups or DLA-DR families: Dw3+Dw4+D1, Dw8+D10, D7+D16+D9, and Dw1. This study provides the basis for DLA-D genotyping at a population level by RFLP analysis. These results also suggest that the genetic organization of the DLA-D region may closely resemble that of the HLA complex.     

The inheritance of restriction fragment length polymorphisms in the flax rust Melampsora lini

Summary Random cDNA sequences synthesized from poly A⁺ RNA extracted from germinated urediospores of the flax rust fungus, Melampsora lini, were used as probes to detect restriction fragment length polymorphisms (RFLPs) in three races of M. lini originating from cultivated flax, Linum usitatissimum, and one race originating from Australian native flax, L. marginale. Fourteen out of 22 probes tested detected RFLPs in the three races from cultivated flax while 19 of the probes detected polymorphisms between these three races and the race from L. marginale. The segregation of seven RFLPs was determined in a family of 19 F₂ progeny derived from a cross between two of the rust races. With six of these the inheritance was consistent, in each case, with the segregation of alleles at a single locus. Inheritance of the seventh was unusual and an explanation involving two loci with null alleles at each was proposed. No linkage was detected between any of the RFLP loci and nine unlinked loci specifying avirulence.     

Heterogeneity of human C4 gene size

In this article we present a study showing that the human C4 genes differ in length because of the presence or absence of a 6.5 kb intron near the 5 end of the gene. DNA from individuals of known HLA, factor B, and C4 haplotypes was analyzed for restriction fragment length polymorphism (RFLP) by Southern blot analysis with C4-specific cDNA probes. The RFLP patterns obtained showed that the C4 genes are either 22.5 kb or 16 kb in length. They are referred to as long and short C4 genes, respectively. A population study was carried out to examine the distribution of the gene size according to C4 allotypes and haplotypes. Long C4 genes included all C4A genes studied and also some C4B allotypes, e. g., B1 on most C4 A3B1 haplotypes. Similarly, C4B null genes were found to be of the long form. Other C4B allotypes tested were found to be coded for by short C4 genes, including B2, B1 in C4 A6B1 and C4 AQOB1 (with a single C4B gene haplotype).Abbreviations used in this paper C4 fourth component of complement - C2 second component of complement - BF factor B - MHC major histocompatibility complex - RFLP restriction fragment length polymorphism - EDTA ethylenediaminetetraacetic acid - SDS lauryl sulfate, sodium salt     

Codominant PCR-based markers for Pinus taeda developed from mapped cDNA clones

 We report a strategy for developing codominant PCR-based genetic markers by using sequenced cDNA clones from loblolly pine (Pinus taeda L.). These clones were previously used as probes for detecting restriction fragment length polymorphisms (RFLPs) to generate linkage maps. After assessing the complexity of banding patterns from Southern blots, we selected clones representing relatively simple gene families, and then determined nucleotide sequences for about 200 bp at each end of the cDNA inserts. Specific PCR primers were designed to amplify samples of genomic DNA derived from two loblolly pine mapping populations. Polymorphisms were detected after digesting the amplified DNA fragments with a battery of restriction endonucleases, and most polymorphisms were inherited in a Mendelian fashion. These newly identified genetic markers are codominant and relatively simple to use. By assaying DNA from individuals used to construct RFLP maps, we show that most of these markers map to the same position as the RFLP loci detected using their corresponding cDNAs as probes, implying that these markers have been converted from RFLP to PCR-based methods. These PCR-based markers will be useful for genome mapping and population genetics. Received: 10 February 1998 / Accepted: 25 February 1998     

TaqI HLA-B and -DRB RFLP analysis can predict disease in siblings of affected children with 21-hydroxylase deficiency

Summary The possibility of using TaqI restriction fragment length polymorphism (RFLP) analysis of the HLA-B locus and the HLA-DR-DQ subregions, flanking the 21-hydroxylase genes, for predicting disease in siblings of children with 21-hydroxylase deficiency was analyzed in 12 nuclear families with at least one affected child and a total of 18 at-risk off-spring. As part of the study allelic TaqI HLA-B RFLP patterns were determined in homozygous cell lines and families. The frequencies of individuals homozygous for TaqI allelic patterns of the different investigated HLA loci, each locus alone and in various combinations, were determined in 100 random controls. In all 12 families it was possible to make correct genetic diagnosis by the use of only one restriction enzyme, TaqI, and two locus-specific HLA cDNA probes, HLA-B and -DRB. In all families four haplotypes were obtained. Thus, affected siblings as well as carriers could be identified. Seven of the eight sibling pairs concordant for 21-hydroxylase deficiency had pairwise identical TaqI HLA-B-DRB-DQA-DQB haplotypes. The last disease-concordant sibling pair had inherited different haplotypes from their mother, who had nonclassical 21-hydroxylase deficiency. None of the ten healthy children shared both haplotypes with their affected sibling(s). Early prenatal suppression of the fetal adrenal cortex with fluorinated corticosteroids can prevent virilization of female fetuses with 21-hydroxylase deficiency. In most cases RFLP analysis of the 21-hydroxylase genes is not informative enough for prenatal diagnosis. Our results from the present retrospective family study indicate that TaqI HLA-B and -DRB RFP analysis will be a valuable tool for first trimester assessment of 21-hydroxylase deficiency. TagI HLA-B and -DRB RFLP analysis can be performed on DNA from chorionic villi biopsies obtained in the 8th week of pregnancy. Supplemented with sex determination, early withdrawal of prophylactic steroid therapy will thus be feasible when the mother carries a male or an unaffected female fetus.     

RFLP analysis and linkage mapping in Solanum tuberosum

Summary A morphologically and agronomically heterogeneous collection of 38 diploid potato lines was analysed for restriction fragment length polymorphisms (RFLPs) with 168 potato probes, including random genomic and cDNA sequences as well as characterized potato genes of known function. The use of four cutter restriction enzymes and a fragment separation range from 250 to 2,000 bases on denaturing polyacrylamide gels allowed the detection of RFLPs of a few nucleotides. With this system, 90% of all probes tested showed useful polymorphism, and 95% of those were polymorphic with two or all three enzymes used. On the average, 80% of the probes were informative in all pairwise comparisons of the 38 lines with a minimum of 49% and a maximum of 95%. The percentage of heterozygosity was determined relative to each other for each line and indicated that direct segregation analysis in F1 populations should be feasible for most combinations. From a backcross involving one pair of the 38 lines, a RFLP linkage map with 141 loci was constructed, covering 690 cMorgan of the Solanum tuberosum genome.     

Organization and evolution of C4 and CYP21 genes in primates: importance of genomic segments

The evolutionary relationship between two central major histocompatibility complex (MHC) genes, C4 and CYP21, was investigated by employing pulsed field gel electrophoresis (PFGE) and conventional restriction fragment length polymorphism (RFLP) analyses in human and nonhuman primates. Using Taq I in conjunction with C4 and CYP21 probes, it has been found that there are four major types of C4 genes [defined by 7.0, 6.4, 6.0, and 5.4 kilobases (kb) Taq I fragments] and two major types of CYP21 genes (3.7 and 3.2 kb fragments) in human and nonhuman primates including chimpanzee, gorilla, and orangutan. All of the eight possible combinations of C4 and CYP21 genes can be identified on one or more human ancestral haplotypes (AH). It is concluded that each of the major types of C4 and CYP21 (and each of the combinations between these) predated human speciation. PFGE analysis with Mlu I and Pvu I suggested that each C4 + CYP21 segment has a specific length of 30–50 kb and that each AH carries one, two, three, or even more segments. In the case of C4, it is important to note that there is no simple relationship between the RFLP and the protein classifications. Thus, at least some of the expressed polymorphisms could be relatively recent in that they are carried by the same or different gene types. These findings are consistent with the hypothesis that M MHC AHs have been formed from a large pool of specific genomic segments and that further haplospecific polymorphism has developed subsequently.     

Isolation of chromosome-21-specific DNA probes and their use in the analysis of nondisjunction in Down syndrome

Summary Thirteen single-copy, chromosome-21-specific DNA probes were isolated from a recombinant library made from flow-sorted chromosome 21 DNA and regionally mapped using a panel of somatic cell hybrids. Five probes mapped in the 21q21-q22.1 region, six to the 21q22.1-qter region, and one to each of the regions 21q22.1-q22.2 and 21q22.3. Two of these probes, one of which maps in the critical region for Down syndrome, have recently been shown to be expressed at high levels in Down syndrome brain tissue (Stefani et al. 1988). Following preliminary screening for restriction fragment lenght polymorphisms (RFLPs), five polymorphisms were discovered with four of the chromosome 21 DNA probes. A frequent MspI polymorphism detected by one of the probes was used in conjunction with four previously described polymorphic chromosome 21 probes to analyse the origin of nondisjunction in 33 families with a child or fetus with trisomy 21. The parental origin of the additional chromosome 21 was determined in 12 cases: in 9 (75%) of these it was derived from the mother and in the other 3 cases (25%) it was of paternal origin. Cytogenetic analysis of Q-banding heteromorphisms was informative in three of five families tested, and in each case the RFLP results were confirmed. The meiotic stage of nondisjunction was defined with confidence in five families, the results being obtained with pericentromeric RFLP or cytogenetic markers. Recombination between two nondisjoined chromosomes was demonstrated in one family and is consistent with the view that a lack of recombination between chromosome 21 homologues or failure of their conjunction is not the invariable cause of trisomy 21.     

Mapping of C2, Bf, and C4 genes to the swine major histocompatibility complex (swine leukocyte antigen)

Three miniature swine lines, inbred for swine leukocyte antigen (SLA) haplotypes, a, c, and d, and a recombinant line, haplotype g, were analyzed for possible restriction fragment length polymorphisms (RFLP) by Southern blot hybridization with human C2, factor B (Bf), and C4 specific probes. The search for RFLP by using a human C2 probe failed to reveal any variants. However, a Taq I polymorphism was identified with the human Bf probe and Bam HI and Pvu II polymorphisms were identified with the human C4 probe. Overlapping restriction fragments were found with the C2 and Bf probes, which strongly suggests close linkage of C2 and Bf genes in swine. Segregation analyses of the Bf and C4 polymorphisms indicated that the polymorphic fragments followed a Mendelian pattern of inheritance. The recombinant haplotype g, which expresses class I genes of haplotype c and class II genes of haplotype d, was shown to produce an identical RFLP pattern, by using the Bf and C4 probes, as haplotype d, but different from that of haplotype c. This indicates that there is a close association of [C4-Bf-C2] and class II genes in miniature swine. Although these data do not show conclusively the location of the [C4-Bf-C2] genes, it is hypothesized that swine [C4-Bf-C2] genes are located between the class II and class I genes, as has been demonstrated in mouse and man.     

Genetic diversity and differentiation of indica and japonica rice detected by RFLP analysis

Summary Genetic diversity and differentiation in indica and japonica groups of the cultivated rice (Oryza sativa L.) were studied by assaying DNA restriction fragment length polymorphisms of 12 indica and 14 japonica rice lines digested with three restriction endonucleases. A total of 49 probes were selected to represent the entire RFLP map at intervals of 20–30 cM. It was shown that 95 of the 145 possible probe/enzyme combinations, involving 43 probes and all three enzymes, detected restriction fragment length variation, and the degree of polymorphism varied greatly from one probe/enzyme combination to another. These results demonstrate that indica rice is genetically more diverse than japonica type. Significant differentiation between the two rice groups was detected by 33 probes representing 11 of the 12 rice chromosomes. It was deduced that the processes leading to differentiation involved a combination of molecular events that include base substitutions and insertion/deletions.     

Genetic linkage mapping in Acacia mangium 1. Evaluation of restriction endonucleases, inheritance of RFLP loci and their conservation across species

Random genomic probes were used to assess levels of restriction fragment length polymorphism (RFLP) in two 2-generation outbred pedigrees of Acacia mangium Willd. Probes were evaluated for their ability to detect polymorphic loci in each pedigree and to determine the relative efficiency of different restriction enzymes in revealing polymorphisms. Sixty two percent of the probes which detected single- or low-copy number sequences revealed polymorphisms with at least one restriction enyzme. HpaII was the most efficient in detecting polymorphism among first-generation individuals. The recognition sequence of HpaII contains a CpG dimer, suggesting that cytosines in the CpG sequence may be hotspots for mutation in plant genomes, as previously reported in bacterial and mammalian genomes. Mendelian inheritance of 230 loci was demonstrated based on single-locus segregation in second-generation individuals. Less than 5% of loci showed evidence of segregation distortion. The proportion of fully informative loci (15%) was lower than previously reported in eucalypts reflecting the lower level of genetic diversity in A. mangium. The RFLP probes are suitable for the construction of a high-density genetic linkage map in A. mangium. Cross-hybridisation of the A.mangium RFLPs to DNA from species representing the three subgenera of the genus Acacia indicates that these markers could be used in breeding programs of other diploid acacias, for comparative studies of genome organisation, and for phylogenetic studies. Received: 5. June 1999 / Accepted: 30 July 1999     

Restriction fragment length polymorphism of DQB and DRB class II genes of the ovine major histocompatibility complex

The ovine major histocompatibility complex (MhcOvar) class II region was investigated by Southern blot hybridizations using ovine probes specific for the second exons of Ovar-DRB and Ovar-DQB genes. Multiple bands were revealed when genomic DNA was digested with each of five restriction enzymes (Bam HI, Eco RI, Hin dIII, PvuII and TaqI), and successively hybridized with the two radiolabeled ovine probes. Restriction fragment length polymorphisms (RFLPs) were analysed in 89 sheep originating from six inbred families and the inheritance of the fragment patterns was determined. Forty-one fragments were recorded with the DQB probe; 32 were detected with the DRB probe. They constituted 9 DQB and 10 DRB allelic patterns. Twelve DQB-DRB haplotypes were resolved in this study.     

Mapped DNA probes from loblolly pine can be used for restriction fragment length polymorphism mapping in other conifers

A high-density genetic map based on restriction fragment length polymorphisms (RFLPs) is being constructed for loblolly pine (Pinus taeda L.). Consequently, a large number of DNA probes from loblolly pine are potentially available for use in other species. We have used some of these DNA probes to detect RFLPs in 12 conifers and an angiosperm. Thirty complementary DNA and two genomic DNA probes from loblolly pine were hybridized to Southern blots containing DNA from five species of Pinus (P. elliottii, P. lambertiana, P. radiata, P. sylvestris, and P. taeda), one species from each of four other genera of Pinaceae (Abies concolor, Larix laricina, Picea abies, and Pseudotsuga menziesii), one species from each of three other families of Coniferales [Sequoia sempervirens (Taxodiaceae), Torreya californica (Taxaceae) and Calocedrus decurrens (Cupressaceae)], and to one angiosperm species (Populus nigra). Results showed that mapped DNA probes from lobolly pine will cross-hybridize to genomic DNA of other species of Pinus and some other genera of the Pinaceae. Only a small proportion of the probes hybridized to genomic DNA from three other families of the Coniferales and the one angiosperm examined. This study demonstrates that mapped DNA probes from loblolly pine can be used to construct RFLP maps for related species, thus enabling the opportunity for comparative genome mapping in conifers.     

Diversity of culturable denitrifying bacteria

Sequence divergence in the ribosomal genes of known strains and isolates of aquatic denitrifying bacteria was investigated using restriction fragment length polymorphism (RFLP) analysis. The same cultures were characterized for their homology with antibody and gene probes for nitrite reductase (NiR), a key enzyme in the denitrification pathway, and for amplification with a set of polymerase chain reaction primers designed to amplify a portion of the NiR gene. The NiR probes were developed from Pseudomonas stutzeri (ATCC 14405) and several P. stutzeri strains were included in the analyses. The RFLP analysis clustered most of the P. stutzeri strains together, but detected considerable diversity within this group. Isolates from three aquatic environments exhibited within —and among — habitat diversity by RFLP. Hybridization with the NiR probes and amplification with the NiR primers were not correlated with the clustering of strains by rDNA RFLP analysis. The relationships among strains deduced from ribosomal DNA RFLP reflect heterogeneity within the P. stutzeri group and among other pseudomonads, and the patterns differ from those inferred from specificity of the NiR probes.Abbreviations NiR Nitrite reductase - PCR polymerase chain reaction - RFLP restriction fragment length polymorphism     

Polymorphisms and gross structure of glycophorin genes in common chimpanzees

Using human α glycophorin cDNA probe and six restriction enzymes, we examined the homologues of human glycophorin genes in genomic DNA of 11 unrelated chimpanzees. We show that, in contrast to the human, the chimpanzee exhibits an unusual array of nonrandomly distributed restriction fragment length polymorphisms (RFLP). No clear correlation was found between the RFLP and the V-A-B-D blood-group phenotypes of the subjects, with one possible exception. However, pairs of allelic RFLP occurring at a relatively high frequency were identified. In addition, the homology of chimpanzee glycophorin genes to the human genes was examined using as probes synthetic oligonucleotides specifying distinct regions of human glycophorin genes. We show that the glycophorin gene family in the chimpanzee consists of at least three members that are homologous to the human α, δ, and E genes (glycophorins A, B, and E) and may share a similar gross structure and overall organization. This research was supported by National Institutes of Health Grant GM 16389.     

An RFLP linkage map for loblolly pine based on a three-generation outbred pedigree

A genetic linkage map for loblolly pine (Pinus taeda L.) was constructed using segregation data from a three-generation outbred pedigree consisting of four grandparents, two parents, and 95 F₂ progeny. The map was based predominantly on restriction fragment length polymorphism (RFLP) loci detected by cDNA probes. Sixty-five cDNA and three genomic DNA probes revealed 90 RFLP loci. Six polymorphic isozyme loci were also scored. One-fourth (24%) of the cDNA probes detected more than 1 segregating locus, an indication that multigene families are common in pines. As many as six alleles were observed at a single segregating locus among grandparents and it was not unusual for the progeny to segregate for three or four alleles per locus. Multipoint linkage analysis placed 73 RFLP and 2 isozyme loci into 20 linkage groups; the remaining 17 RFLP and 4 isozyme loci were unlinked. The mapped RFLP probes provide a new set of codominant markers for genetic analyses in loblolly pine.