Polymorphisms within the HLA-DRw6 haplotype

We have analyzed the HLA class II gene products from HLA-DRw6 homozygous cells. Epstein-Barr virus-transformed B-cell lines were internally labeled with [³⁵S]-methionine. An NP-40 lysate of the cells was subjected to immunoprecipitation, first with a DRw52-like-specific monoclonal antibody and subsequently with a DR-specific framework antibody. The DR region-encoded gene products were analyzed by one-dimensional gel isoelectric focusing and two-dimensional gel electrophoresis. It is shown that DRw6 homozygous cell lines contain at least two nonallelic DR chains, one carrying a DRw52 determinant and one DRw52-negative population. Both chains appear to be polymorphic between the cellularly defined subtypes of DRw6. The determinant responsible for the differential mixed lymphocyte culture reactivity of Dw18 and Dw19 cells resides on the DRw52-positive population, whereas the Dw6-Dw9 differences are attributed to determinants on both populations of DR light chains. The Dw16-derived DRw52⁺ chain much resembles the Dw18 DRw52⁺ light chain whereas there is a clear-cut difference between these two subtypes in the DRw52^– population. We conclude that, for DRw6 homozygous cells, the cellularly recognized D determinants are probably located on DR-encoded molecules, both DRw52⁺ and DRw52^–, and that charge shift of these chains is at least partly responsible for differential recognition of these cells in mixed lymphocyte cultures.Abbreviations used in this paper: MLC mixed lymphocyte culture - 1D-IEF one-dimensional isoelectric focusing - 2D two-dimensional - moab monoclonal antibody     

Restriction fragment length polymorphism evaluation of six peanut species within the Avachis section

Summary Restriction fragment length polymorphisms (RFLP) were assessed among accessions within six peanut species of the Arachis section: tetraploid cultivated species, A. hypogaea; tetraploid wild species, A. monticola; and four diploid wild species, A. batizocoi,A. cardenasii, A. duranensis and A. glandulifera. While the two tetraploid species did not show polymorphism with 16 PstI-generated random genomic probes, two of seven seed cDNA probes detected polymorphisms. The RFLP variation detected by two seed cDNA probes appeared to be related to structural changes occurring within tetraploid species. The botanical var. fastigiata (Valencia market type) of A. hypogaea subspecies fastigiata was shown to be the most variable. Arachis monticola was found to be more closely related to A. hypogaea subspecies hypogaea than to subspecies fastigiata. Diploid species A. cardenasii, A. duranensis, and A. glandulifera showed considerable intraspecific genetic diversity, but A. batizocoi showed little polymorphism. The genetic distance between the cultivated peanut and wild diploid species was found to be closest for A. duranensis.Florida Agricultural Experiment Station, Journal Series No. R-01493     

Restriction fragment length polymorphism and molecular taxonomy in Vitis vinifera L.

Forty-six accessions of grapevine (V. vinifera L.) were compared by restriction fragment length polmorphism (RFLP) analysis, and 111 informative or unique restriction fragments were found that revealed an important level of polymorphism. RFLP patterns were compared in two ways: by calculating electrophoretic similarity degree values further analyzed by principal component analysis and by studying the distribution of rare restriction fragments. Six taxonomic groups could be defined, which partially confirmed relationships derived from ampelographical data. Our data support the existence of ecogeographical groups.     

Restriction fragment length polymorphism maps and the concept of graphical genotypes

Summary With the advent of high density restriction fragment length polymorphism (RFLP) maps, it has become possible to determine the genotype of an individual at many genetic loci simultaneously. Often, such RFLP data are expressed as long strings of numbers or letters indicating the genotype for each locus analyzed. In this form, RFLP data can be difficult to interpret or utilize without complex statistical analysis. By contrast, numerical genotype data can also be expressed in a more useful, graphical form, known as a graphical genotype, which is described in detail in this paper. Ideally, a graphical genotype portrays the parental origin and allelic composition throughout the entire genome, yet is simple to comprehend and utilize. In order to demonstrate the usefulness of this concept, graphical genotypes for individuals from backcross and F2 populations in tomato are described. The concept can also be utilized in more complex mating schemes involving two or more parents. A model that predicts the accuracy of graphical genotypes is presented for hypothetical RFLP maps of varying marker spacing. This model indicates that graphical genotypes can be more than 99% correct in describing a genome of total size, 1000 cM, with RFLP markers located every 10 cM. In order to facilitate the application of graphical genotypes to genetics and breeding, we have developed computer software that generates and manipulates graphical genotypes. The concept of graphical genotypes should be useful in whole genome selection for polygenic traits in plant and animal breeding programs and in the diagnosis of heterogenously based genetic diseases in humans.     

Restriction fragment length polymorphisms of three collagen genes in Russians and Buryats.

The restriction fragment length polymorphisms (RFLPs) of 3 collagen genes have been studied in two USSR populations. Four high-frequency alleles, also reported in other populations, were observed. The frequencies of alleles for the polymorphic sites MspI and EcoRI of the COL1A2 gene, HindIII of the COL2A1 gene and EcoRI of the COL3A1 gene were found to be 0.836, 0.398, 0.447 and 0.786, respectively, in Russians from Moscow (Caucasians), and 0.883, 0.519, 0.648 and 0.809, respectively, in Buryats from the Baikal region (Mongoloids). The RFLPs showed a fair agreement with the Hardy-Weinberg equilibrium. The frequencies were compared with those of other populations.     

Restriction fragment length polymorphism of the Vibrio anguillarum serovar O1 virulence plasmid.

Seventy-eight strains of Vibrio anguillarum serovar O1, all harboring one 65- to 70-kilobase plasmid, were typed according to restriction fragment length polymorphism of the plasmid. Six types, three of which comprised 96% of the strains examined, were produced with the restriction endonuclease BamHI. The fragment length polymorphism type did not correlate to any of 12 different phenotypic properties tested.     

Restriction fragment length polymorphisms in the mushrooms Agaricus brunnescens and Agaricus bitorquis

Two Agaricus species, A. brunnescens (a commercial mushroom) and A. bitorquis (a wild, edible species), were examined for restriction fragment length polymorphisms. EcoRI-digested nuclear DNA from isolates of both species were cloned in plasmid vector pUC18. Ten random recombinant clones were used in Southern DNA-DNA hybridizations to probe EcoRI-digested DNA from 11 A. brunnescens isolates (7 commercial, 2 wild type, and 2 homokaryotic) and 7 A. bitorquis isolates. Most cloned fragments were polymorphic in both species. There were fewer different genotypes than expected, however, in the sample of commercial A. brunnescens strains. DNA from homokaryotic strains showed fewer bands in most hybridizations than DNA from heterokaryotic strains. All A. bitorquis isolates could be distinguished from each other as well as from every A. brunnescens strain. Putative homokaryons were detected by the loss of polymorphic bands among protoplast regenerates from one commercial strain and two strains collected in the wild.     

Restriction fragment length polymorphism of cardiac myosin heavy chain gene in rats and its strain distribution

The distribution of an RFLP in EcoRI fragments of the cardiac myosin heavy chain gene among 29 strains of laboratory rats was examined. Southern blot hybridization of rat genomic DNAs with rat cardiac myosin heavy chain cDNA as a probe demonstrated an interstrain variation in one of eight EcoRI fragments. Of the 28 inbred strains examined, 10 had a fragment of 10 kbp, whereas 18 had a fragment of 7.5 kbp. The 15 samples of the remaining strain (Iar: WI outbred stock) had fragments of either 7.5 kbp or 10 and 7.5 kbp, indicating that this strain has maintained heterogeneity of these fragments.     

Restriction fragment length polymorphism caused by a deletion involving Alu sequences within the human alpha 2-plasmin inhibitor gene

A restriction fragment length polymorphism within the human alpha 2-plasmin inhibitor gene has been detected by Southern blot hybridization using an alpha 2-plasmin inhibitor cDNA probe. This restriction fragment length polymorphism can be attributed to the presence of two alleles, A and B, that are distributed in Hardy-Weinberg equilibrium with frequencies of 73.5% and 2.65%, respectively, in 66 unrelated Caucasian individuals or with frequencies of 51.0% and 49.0%, respectively, in 50 unrelated Japanese individuals. The minor allele, B, is due to a deletion of about 720 base pairs in intron 8 of the alpha 2-plasmin inhibitor gene. Sequence analysis of the deletion junction in allele B and the corresponding regions of allele A demonstrated the presence of oppositely oriented Alu sequences at the 5' and 3' deletion boundaries. These data suggest that this restriction fragment length polymorphism was caused by intrastrand recombination between Alu sequences.     

Restriction fragment length polymorphism of the rat albumin gene in Sprague-Dawley rats and its application in genetic study of analbuminemia.

Restriction fragment length polymorphism of the rat albumin gene was discovered in a stock of Sprague-Dawley rats by Southern blots of rat liver DNAs using cloned albumin cDNA, prAlb-1 (1), as a probe. The polymorphic DNA fragments were observed when rat DNAs were digested with either Hind III or Pst 1 and the difference in length of the DNA fragments in Hind III or Pst 1 digests was estimated as 1.4 kbp. When DNAs were digested with EcoR I, restriction fragment length polymorphism was not observed. Therefore, this polymorphic DNA was concluded to be located in the flanking sequence. Structural analysis of the cloned albumin gene showed that the polymorphism was located in the 3'-flanking sequence. With this polymorphism as a marker of the albumin structural gene, the phenotype of analbuminemia, which is an autosomally recessive trait, was found to be linked to the structural gene of albumin.     

Restriction fragment length polymorphism segregation analysis of the Li locus in Trifolium repens L.

The Li locus in white clover controls the presence of cyanogenic -glucosidase (linamarase) activity in leaf tissue, such that plants homozygous for the null allele (li) have no linamarase activity in this tissue. The isolation of a cDNA clone from linamarase mRNA is described. The cDNA clone is used to further characterise alleles of the Li locus. Northern blot analysis shows that plants homozygous for the null allele (li li) produce very reduced levels of mRNA which hybridises to the cDNA. Heterozygous plants (Li li), which have intermediate levels of enzyme activity, produce intermediate levels of mRNA. Southern blot analysis of Hind III digested genomic DNA shows that the white clover genome contains three genes with homology to the linamarase cDNA and that at least two of these genes segregate independently. Analysis of the cosegregation of linamarase activity and the presence of genomic restriction fragments identifies the genomic sequence specifying linamarase structure and indicates either a structural or cis acting control function of the Li locus.     

Restriction maps and restriction fragment length polymorphisms of the human 21-hydroxylase genes

Restriction maps were constructed for the two human 21-hydroxylase genes (21-OHA and 21-OHB) by using DNA from subjects homozygous for a deletion of each gene. Comparing the patterns of these two genes, a KpnI restriction site occurred in the 21-OHA gene in place of a TaqI site in the 21-OHB gene about 1-kb from the 5' end of the gene, and an extra EcoRI site was located 500 bp 5' to the common EcoRI site. The DNA of fourteen unrelated normal subjects was digested with nine restriction endonucleases (AccI, BamHI, BgIII, EcoRI, HindIII, KpnI, MspI, SacI and TaqI). Restriction fragment length polymorphisms were found with EcoRI, HindIII and AccI that resulted from polymorphic endonuclease sites outside the genes.     

Restriction fragment length polymorphism analysis of CCDD genome species of the genus Oryza L.

Restriction fragment length polymorphisms (RFLPs) were studied in fourteen accessions of CCDD genome allotetraploid wild rice species (Oryza latifolia, O. alta and O. grandiglumis). Fourteen nuclear RFLP markers previously mapped in AA genome-cultivated rice were used as probes. A phylogenetic tree, constructed by parsimony analysis based on RFLPs, grouped the accessions according to their geographic origin from Central or South America. Oryza alta, O. grandiglumis and one accession of O. latifolia grouped together as a subgroup, and our results suggested that the three taxa should be considered as populations of a single complex species. Duplicate loci, representing the two constituent genomes of the allotetraploid, were observed for most RFLP markers. By comparing RFLPs from the allotetraploids with those from a CC genome diploid wild species (O. officinalis), it was possible to detect RFLPs specific for both the CC and DD genomes of the allotetraploid. In inter-accession F2 populations, independent segregation of RFLP markers for CC and DD genomes was observed.     

Restriction fragment length polymorphism analysis of Cryptosporidium parvum isolates of bovine and human origin.

Cryptosporidium parvum oocysts isolated from different hosts and geographical areas were compared by restriction endonuclease analysis of repetitive DNA: Iowa (bovine), Florida (bovine), New York (bovine), Peru (human), Brazil (human), and Mexico (human). Southern blot hybridization analysis was performed using the restriction endonuclease enzyme Eco RI and the DNA probe pV47-2. The probe hybridized with 18 bands present in all the isolates. The Brazilian, Mexican, and Peruvian human isolates had an additional common band of 4.3 kbp that was absent in the bovine isolates. Two extra bands of 14 and 12 kbp were present in the Brazilian isolate whereas the Mexican isolate had an extra band of 14 kbp. When the Iowa and Peru C. parvum isolates were passed twice through calves, oocysts recovered from both passages showed identical banding patterns, suggesting that recombination of the repetitive sequences was not altered during sexual reproduction. The DNA digested with other restriction endonucleases were tested confirming differences between isolates. A genomic DNA library is currently being produced to better define isolate variation in C. parvum.     

Restriction fragment length polymorphism (RFLP) analysis in wheat. I. Genomic DNA library construction and RFLP analysis in common wheat

To develop detailed linkage maps of restriction fragment length polymorphism (RFLP) sites in wheat chromosomes, it was necessary to construct a genomic DNA library and to characterize the clones obtained. Forty-nine per cent of the clones were of single or low copy number per genome. With 91 clones of this class, as probes, and with two to four restriction endonucleases, for DNA digestion, RFLPs were examined among eight common wheats and a single emmer wheat. About 20% of the probes, and 13% of the probe-enzyme combinations revealed genetic polymorphism among the common wheats. DNA deletions account for most of the genetic differences among these wheat genomes. Based on the RFLP data, phylogenetic distances among the nine polyploid wheats were estimated, and a dendrogram showing the genetic relationships among them was constructed.     

Restriction fragment length polymorphism and evolution of the mouse immunoglobulin constant region gamma loci

Genomic DNA from twelve laboratory mouse strains, in addition to 21 wild-derived strains belonging to different taxa (Mus musculus domesticus, Mus musculus musculus, Mus spretus, Mus macedonicus, a and Mus spicilegus) and four mouse strains that are evolutionarily more distant, were analyzed by Southern blot for polymorphism of the Ig heavy chain constant region isotype (Igh-C) and for the distribution of the duplicated Igh-1 (C2) haplotype. Distinct allelic forms of each Igh-C locus could be defined by restriction fragment length polymorphism (RFLP). In laboratory mouse strains RFLP proved to be more sensitive in the detection of Igh-4 (C1) alleles than serological methods. Taq I digestion allowed the definition of two alleles in the Igh-8 (C3) locus, which is absolutely conserved at the protein levels. More extensive RFLP could be found in wild strains belonging to the subgenus Mus and in the evolutionarily more distant Mus species belonging to other subgenera. In previous studies we have shown that the Igh-1 locus is duplicated in M. m. musculus subspecies. We now extend this observations to the wild mouse strains belonging to M. spicilegus and M. macedonicus species and to the evolutionarily more distant wild mouse strain Mus pahari (subgenus coelomys), which is thought to have diverged from domestic mice about 5 million years ago. In addition, we found a similar RFLP pattern in ten of 18 wild mice trapped in India, suggesting that the haplotype containing the two Igh-1-like genes, organized in tandem as distinct isotypes, is widely spread in natural populations. The evolution of murine Igh-C-encoded isotypes is also discussed. Correspondence to: P.-A. Cazenave.