Restriction fragment length polymorphisms in bovine major histocompatibility complex class II ß-chain genes using bovine exon-containing hybridization probes

Restriction fragment length polymorphisms (RFLPs) have been identified in the bovine MHC class II region using five hybridization probes constructed from two bovine genomic clones. Four probes were constructed from a bovine DR beta-like gene, BoLA-DRB2. These included a probe containing the complete beta 1 exon (R2-beta 1), a probe containing the last 129 base pairs of the beta 2 exon (R2-beta 2), a probe containing intron immediately 5' of the beta 2 exon (R2-5' beta 2), and a probe containing the complete transmembrane exon (R2-TM). A fifth probe was constructed from a novel bovine beta-chain gene, BoLA-DIB, and contained the entire TM exon (I1-TM). R2-beta 1 defined very little polymorphism. R2-beta 2 hybridized to several fragments but one or two fragments hybridized much stronger on all Southern blots and it was presumed these corresponded to BoLA-DRB2 fragments. By using R2-5' beta 2 as a probe, these BoLA-DRB2 fragments were confirmed: 6.4 and 2.7-kb Eco RI alleles, 1.7- and 1.5-kb Pvu II alleles, 5.9-, 5.4-, 3.7- and 1.9-kb TaqI alleles, and a non-polymorphic 22.5-kb BamHI fragment. I1-TM identified three alleles with TaqI. To investigate the linkage between the RFLP alleles, 166 offspring of five sires were tested. Complete linkage was found for all RFLPs identified with the BoLA-DRB2 probes. However, the RFLP patterns of 13 calves out of 58 indicated recombination between BoLA-DRB2 and BoLA-DIB.     

Mucopolysaccharidosis IVA: structural gene alterations identified by Southern blot analysis and identification of racial differences

Ninety-six alleles (36 alleles of Japanese and 60 of Caucasian origin) from forty-eight patients with mucopolysaccharidosis IVA were investigated for structural gene alterations using Southern blot analysis. All patients had a previously demonstrated deficiency of N-acetylgalactosamine-6-sulfate-sulfatase and exhibited a wide spectrum of clinical severity. Initially, using the fulllength cDNA as a probe, five of 36 chromosomes from the Japanese patients revealed similar rearrangements with respect to DNA digested with BamHI, SacI, and XhoI. Subsequent analysis using seven genomic fragments, covering the entire gene, enhanced the detection of aberrant fragments produced by the above restriction enzymes. Conversely, the 60 chromosomes of Caucasian origin revealed no evidence of large structural rearrangements when analyzed by these methods. There was a statistically significant difference between the two populations (P < 0.01). A severely affected Japanese patient showed structural rearrangements on both chromosomes by means of BamHI blots. An 8.0-kb fragment and a highly polymorphic 7.0-kb to 11.0-kb fragment present in normal individuals disappeared and two aberrant fragments of 11.5 kb and 12.0 kb were observed. Three other Japanese patients also showed these two aberrant fragments, in addition to the normal fragment pattern, and were thus heterozygous for this rearrangement. Interpretation of Southern blots was difficult because of the complexity of polymorphic bands resulting from variable number of tandem repeat elements. However, by utilizing these aberrant fragments or polymorphic bands, carrier detection was effective, even in families with poorly characterized mutations. Hybridization with probe MG-A (5end genomic probe in intron 1) showed a 8.4-kb fragment in BamHI blots of one Japanese and one Caucasian patient; XhoI, SacI, and EcoRI blots were normal. Since this BamHI alteration was also observed in one normal control, it appears to be a rare nonpathological polymorphism.     

Molecular genetic analysis of the 17p11.2 region in patients with hereditary neuropathy with liability to pressure palsies (HNPP)

Hereditary neuropathy with liability to pressure palsies (HNPP) is in most cases associated with an interstitial deletion of the same 1.5-Mb region at 17p11.2 that is duplicated in Charcot-Marie-Tooth type 1A (CMT1A) patients. Unequal crossing-over following misalignment at flanking repeat sequences (CMT1A-REP), either leads to tandem duplication in CMT1A patients or deletion in HNPP patients. With the use of polymorphic DNA markers located within the CMT1A/HNPP duplication/deletion region we detected the HNPP deletion in 16 unrelated HNPP patients, 11 of Belgian and 5 of French origin. In all cases, the 1.5-Mb size of the HNPP deletion was confirmed by EcoRI dosage analysis using a CMT1A-REP probe. In the 16 HNPP patients, the same 370/320-kb EagI deletion-junction fragments were detected with pulsed field gel electrophoresis (PFGE), while in CMT1A patients, a 150-kb EagI duplication-junction fragment was seen. Thus, PFGE analysis of EagI-digested DNA with a CMT1A-REP probe allows direct detection of the HNPP deletion or the CMT1A duplication for DNA diagnostic purposes.     

Polymorphism in Brucella spp. due to highly repeated DNA.

The species of Brucella are very closely related, but Brucella ovis does not express detectable amounts of a protein, designated BCSP31, that is common to the other species. We studied the lack of expression of BCSP31 by Southern analysis. DNAs from the B. ovis culture collection strains and field isolates were probed with a 1.3-kb HindIII fragment encoding BCSP31 of Brucella abortus. The probe hybridized to a 1.6-kb HindIII fragment of all B. ovis strains tested, showing that the gene is present in B. ovis but occurs on a larger restriction fragment. DNA linkage studies and restriction mapping of the cloned polymorphic region of B. ovis showed that the polymorphism was due to a DNA insertion of approximately 0.9 kb at a site downstream of the BCSP31-coding region. When the 1.6-kb polymorphic B. ovis fragment was used to probe a HindIII Southern blot of cellular DNA of strains of B. ovis and of B. abortus, at least 24 fragments of B. ovis and 6 fragments of B. abortus hybridized to the inserted DNA. Specimens of B. ovis collected over a 30-year period on two continents had similar hybridization patterns. The large difference between B. ovis and B. abortus in the number of copies of the repeated DNA is interesting in the context of the closeness of the Brucella species.     

The glycine cleavage system: structure of a cDNA encoding human H-protein, and partial characterization of its gene in patients with hyperglycinemias.

We have isolated a 1,192-base-long cDNA which encodes the entire structure of a precursor form of human H-protein. The tentatively calculated number of copies for this cDNA appeared to be about four times as many as that of the antithrombin III gene specified by a single locus in the human haploid genome. Southern analysis using H-protein cDNA probe demonstrates a deletion of the 5.0-kb SacI fragment in the genome of a patient with an atypical nonketotic hyperglycinemia in whom there was an inactive H-protein. This SacI fragment was also deleted from the genome of one of seven patients with nonketotic hyperglycinemia resulting from the lesion of glycine decarboxylase. The remaining six patients had common aberrations identified with the 5.2-kb EcoRI and 5.5-kb SacI fragments. Although implication of these defective fragments in pathogenesis is unclear at present, it is suggested that rearrangements occur in multiple genomic loci of patients with nonketotic hyperglycinemia and that this H-protein cDNA can be used for carrier screening.     

DNA restriction-site polymorphisms associated with the alpha 1-antitrypsin gene.

Restriction-site variation in and around the alpha 1-antitrypsin gene has been studied using two genomic probes. With use of restriction enzymes SstI, MspI, and AvaII, three polymorphic sites have been described with a 4.6-kb probe in the 5' portion of the gene. With use of a 6.5-kb probe, polymorphisms in the coding and 3' regions of the gene have been detected with AvaII, MaeIII, and TaqI. All of these polymorphisms are of sufficiently high frequency to be useful in genetic mapping studies. The polymorphisms with AvaII and MaeIII (6.5-kb probe) are particularly useful for prenatal diagnosis. PI types and M subtypes tend to be associated with specific DNA haplotypes; there are two different types of DNA haplotypes associated with PI M1. The extent of linkage disequilibrium differs throughout the region of the alpha 1-antitrypsin gene.     

Polymorphic restriction sites in the horse (β-globin gene cluster

Horse DNA samples digested with PstI and probed with the rabbit beta 1 globin gene show three phenotypes determined by one fragment of variable length (about 5.1 or 3.3 kb). Family data demonstrate that these fragments segregate as Mendelian alleles. The frequencies of the two alleles are 0.66 for the 3.3-kb fragment and 0.34 for the 5.1-kb one. Another polymorphism has been detected with BamHI. Again three phenotypes determined by two alleles (fragments of 7.5 and 3.8 kb) have been observed. Allelic frequencies of the 7.5- and 3.8-kb fragments are 0.24 and 0.76 respectively. The two polymorphic sites are non-randomly associated.     

Mucopolysaccharidosis type II (Hunter disease): 13 gene mutations in 52 Japanese patients and carrier detection in four families

Southern blot analysis of the iduronate sulfatase (IDS) gene in 52 unrelated Japanese patients with mucopolysaccharidosis type II was carried out using a cDNA probe, and mutations in 13 patients (25%) were identified. Of these, 3 had partial gene deletions (in 2 the normal 9.4-kb fragment was absent and in 1 the normal 7.4-kb fragment was absent, as determined by Southern blot analysis using EcoRI-digested DNA, respectively), 2 had gene insertions (in 1 there was a unique 11.2kb fragment and in the other there was a unique 5kb fragment, determined by Southern blot analysis using EcoRI digested DNA), and 8 had rearrangements (in 6 the normal 9.4kb and 7.0kb fragments were absent and a unique 11.2kb fragment was present; in the remaining 2 patients there were different rearrangements). In these 13 patients, the similar Southern blot patterns were indicative of structural alterations of the IDS gene, as revealed when their DNA was digested with HindIII or PstI and probed with IDS cDNA. All patients with these structural alterations were in a clinically severe state, except for 1 with an intermediate clinical phenotype. Our analyses of four families among those of the 13 patients revealed that all four mothers were carriers. The detection of structural abnormalities led to a precise identification of Hunter heterozygotes and revealed one de novo rearrangement in a germ cell of one of the maternal grandparents.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

One of the two genomic copies of the glycine decarboxylase cDNA has been deleted at a 5' region in a patient with nonketotic hyperglycinemia

One of eight patients with nonketotic hyperglycinemia resulted by the lesion in glycine decarboxylase showed the deletion of 0.6-kb SacI and 1.5-kb PstI fragments identified by the cDNA for this protein. A genomic clone, lambda HGDG10, encodes a 5' region of this cDNA in an organized structure and can produce these two fragments. The other clone, lambda HGDG8, carries a processed gene. Southern analysis using a limited segment of this cDNA demonstrated that the 1.7-kb and 1.5-kb PstI fragments predicted from its recognition sites in both genomic clones occur actually in the human genome, indicating that at least two copies of glycine decarboxylase cDNA exist in the haploid genome, and the patient has the glycine decarboxylase gene deleted at a 5' region.     

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.     

Genomic DNA encoding rabbit T cell receptor beta-chains: isotypes and allotypes of C beta

We have discovered sequence differences in DNA encoding the first exon of rabbit T cell receptor beta-chains from unrelated rabbits that probably reflect allelic C beta 1 allotypes. Rabbit I was from a colony bred to maintain the K1-expression mutation Basilea, and rabbit II was from a colony bred to maintain the K1b9 allotype. Genomic DNA from rabbits I and II also exhibit restriction fragment length polymorphism of C beta on Southern blots. In addition, several different restriction enzyme digests of DNA from rabbit I give three bands, whereas DNA from rabbit II gives two when probed with C beta. An approximately 14-kb cloned genomic DNA fragment from rabbit I has two copies of C beta exon 1 and a 6-kb fragment has a third copy, suggesting that rabbit I has three different C beta genes. The DNA sequence of a germ-line genomic DNA fragment encoding the first exon of the beta-chain constant region from rabbit I also has an open reading frame encoding 140 amino acids immediately 5' of the C beta sequence. A corresponding sequence had previously been found in a cDNA clone from the second rabbit (rabbit II).     

Hypobetalipoproteinemia due to an apolipoprotein B gene exon 21 deletion derived by Alu-Alu recombination

We report the molecular defect in an individual with homozygous hypobetalipoproteinemia. A unique TaqI restriction fragment length polymorphism was found in the midportion of the apolipoprotein B (apoB) gene using the genomic probe, pB51. The probe, which identifies TaqI fragments of 8.4 and 2.8 kilobases (kb) in normal individuals, hybridized to a single 11-kb fragment in the proband. The parents of the proband showed all three TaqI fragments, implying that they are heterozygotes for the mutant apoB allele. In this family, the mutant allele cosegregated with low total cholesterol levels and formal linkage analysis gave a decimal logarithm of the ratio score of 3.3 at a recombination frequency of 0. The polymorphic TaqI site was localized to an EcoRI fragment of 4 kb in normal individuals. The corresponding fragment in the proband was 3.4 kb, suggesting a 0.6-kb deletion in the mutant allele. Both the normal 4-kb EcoRI fragment and the mutant 3.4-kb EcoRI fragment were cloned and sequenced. In the normal allele, the 4-kb EcoRI fragment extends from intron 20 to 23. Exon 21 is flanked by Alu sequences that are in the same orientation. The mutant allele had a 694-bp deletion in this region which included a small part of the Alu sequence in intron 20, the entire exon 21, and most of the Alu sequence in intron 21. The polymorphic TaqI site, which lies within the Alu sequence in intron 21, was absent in the proband as a result of the deletion. The deletion of exon 21 results in a frame shift mutation and the introduction of a stop codon. Translation of the encoded mRNA would yield a prematurely terminated protein. This mutant apoB protein would be 1085 amino acids long with the 73 carboxyl-terminal residues out of frame. We postulate that the deletion of exon 21 is the consequence of a crossover event between the Alu sequences in introns 20 and 21 resulting in nonreciprocal exchange between two chromosomes.     

Southern blot analysis with synthetic oligonucleotides. Application to prostatic protein genes.

1. An ethanol precipitation procedure was developed to purify radiolabeled DNA and oligonucleotide probes to be used in Southern blots. 2. The radiolabeled probes produced strong hybridization signals on a clear background on Southern blot analysis of single gene copies even after 5 days of exposure on X-ray films. 3. An oligonucleotide probe complementary to human glandular kallikrein-1 coding region (amino acids 161-167) detected a single DNA fragment after digestion with Bam H1, Hind III or Pst 1. 4. Another oligonucleotide probe coding for the same region of human prostate-specific antigen detected 3 DNA fragments on Southern blots by contrast to a 1.5 kb full length cDNA probe which detected the presence of only one strong hybridization signal. 5. Oligonucleotide probes appear to be excellent tools for gene mapping. Their sensitivity, specificity and limitations can be compared to the one of monoclonal antibodies used in epitope mapping of proteins.     

Polymorphic class II sequences linked to the rat major histocompatibility complex (RT1) homologous to human DR and DQ sequences

Until recently, the analysis of Class II genes linked to the rat major histocompatibility complex, RT1, has been confined to serologic and electrophoretic analysis of their gene products. To obtain a more definitive estimate of the number and relative polymorphism of RT1 Class II sequences, we performed Southern blot analysis of rat genomic DNA employing human cDNA probes specific for Class II heavy and light chain genes. Southern blots of EcoRI and BamHI digests of genomic DNA from ten inbred strains, expressing eight RT1 haplotypes, were hybridized with the human DQ beta or DR beta cDNA that are homologous to Class II light chain sequences. Four to eight bands were observed to hybridize with the light chain cDNA: band sizes ranged from 2.5 to 28 kb. Restriction fragment patterns were polymorphic; the only identical patterns observed were those associated with RT1 haplotypes with identical RT1.B regions. The number and size of bands hybridizing with DQ beta and DR beta suggested a minimum of four light chain sequences in each haplotype. Southern blots of BamHI and EcoRI digests of genomic DNA from the same strains were hybridized with a DR alpha cDNA that is homologous to Class II heavy chain sequences. All RT1 haplotypes expressed either a 10.0-kb or 13.0-kb band when digested with BamHI, and either a 17-kb or 3.7-kb band when digested with EcoRI. Considerably less polymorphism was detected with the DR alpha probe; this observation is consistent with previously reported limited protein polymorphism of the rat equivalent of the I-E alpha subunit. The size and number of bands hybridizing with the DR alpha probe suggests a minimum of two heavy chain sequences. These observations suggest that the RT1 complex includes more Class II sequences than have been observed in serologic and electrophoretic analyses of Class II gene products. Furthermore, the level of polymorphism of RT1 Class II sequences appears to be comparable with mouse and human Class II sequences.     

The organisation of chromatin loops: characterization of a scaffold attachment site

Previous experiments have identified a 657-bp restriction fragment in the non-transcribed region of the Drosophila histone gene cluster that is specifically associated with the histone-depleted nuclear scaffold. The remaining fragments of the 5-kb histone repeat were shown to be readily released from the scaffold; hence it was proposed that the tandemly repeated cluster of histone genes forms a series of 5-kb loops restrained by a nuclear substructure at the sites of attachment. Here we show that the attachment fragment is tightly associated with protease-sensitive material, whereas the solubilized fragments are relatively protein-free. Exonuclease III digestion has been used to map the location of protein complexes on the attachment fragment. We have defined two regions of ~200 bp whose borders provide kinetic barriers to exonuclease III degradation. They are separated by a nucleaseaccessible region of ~100 bp. The protected regions are sufficient to mediate association of the fragment with the histonedepleted nuclei. Sequence analysis reveals an enrichment for sequences closely related to the topoisomerase II cleavage consensus in these two domains.     

Integration and excision of pMC7105 in Pseudomonas syringae pv. phaseolicola: involvement of repetitive sequences.

The site for integration of pMC7105 into the chromosome of Pseudomonas syringae pv. phaseolicola has been mapped to a 2.6-kilobase-pair (kb) Bg/II-EcoRI fragment on this 150-kb indigenous plasmid. Selected excision plasmids resulting from imprecise excision of pMC7105 were used to identify one of the plasmid-chromosome juncture fragments and to characterize the mechanism of recombination from the chromosome. A 14.2-kb BamHI plasmid-chromosome juncture fragment has been identified in pEX8060 (234 kb), an excision plasmid which carries approximately 90 kb of chromosomal sequences to the left of the site of integration. This fragment contains a portion of the 2.6-kb Bg/II-EcoRI fragment as well as chromosomal sequences. Blot hybridization with a probe made from selected fragments of pMC7105 revealed three distinct repetitive sequences, RS-I, RS-II, and RS-III, on this plasmid. The 2.6-kb fragment, to which the site of integration maps, also contains RS-II. Five copies of RS-II are present in pMC7105, and more than 20 copies are present in the chromosome. Eight small excision plasmids were shown to result from recombination among fragments of pMC7105 that contain common repetitive sequences. The results indicate that integration and excision of pMC7105 occur through general recombination at homologous repetitive sequences.     

Localization of flagellin genes on the physical map of Methanococcus voltae

Methanococcus voltae DNA, digested individually with the restriction enzymes ApaI, SacII, BamHI, or EagI, was resolved by pulsed-field gel electrophoresis reproducing the previously published digestion patterns. Hybridization of a flagellin gene-specific probe to such gels dried down (unblots) resulted in the identification of one band per enzyme harboring the flagellin genes. These bands all overlapped, revealing that an approximately 15-kb BamHI/EagI DNA fragment should harbor the flagellin genes. Double digestion with BamHI and EagI resulted in the resolution of two bands in the 15-kb region of the gel. Separation of these two fragments prior to blotting and probing with a flagellar gene-specific probe revealed that one of these fragments possessed the flagellar sequences. The presence of an EagI restriction site in flaB3 localized the flagellin genes precisely at the junction of EagI fragments Ea2 and Ea5 at approximately the 1800-kb position of the physical map.