Satellite-tagged transcribing sequences in Bubalus bubalis genome undergo programmed modulation in meiocytes: possible implications for transcriptional inactivation.

We cloned and sequenced a 1378 bp BamHI satellite DNA fraction from the water buffalo Bubalus bubalis and have studied its expression in different tissues. The GC-rich sequences of the resultant contig pDS5 crosshybridize only with bovid DNA and are not conserved evolutionarily. Typing of buffalo genomic DNA using pDS5 with several restriction enzymes revealed multilocus monomorphic bands. Similar typing of cattle, buffalo, goat, sheep, and gaur genomic DNA revealed variations in copy number and allele length giving rise to species-specific band patterns. Expression study of pDS5 in bubaline samples by RNA slot-blot, Northern blot, and RT-PCR showed various levels of signal in all the somatic tissues and germline cells except heart. A GenBank database search revealed homology of pDS5 sequences in the 5' region from nt 1-1261 with collagen gene. An AluI typing analysis of DNA from bubaline semen samples showed consistent loss of two bands. The presence of corresponding bands in somatic tissues suggests a sequence modulation within the pDS5 array in meiocytes during spermatogenesis, which is restored in the somatic cells after fertilization. Modulation of the satellite-tagged transcribing sequence in the meiocytes may be a mechanism of its inactivation.     

Somatic cell mapping of conglutinin (CGN1) to cattle syntenic group U29 and fluorescence in situ localization to Chromosome 28

A 260-bp genomic PstI fragment, which encodes a portion of the carbohydrate recognition domain, was used along with hybrid somatic cells to map the conglutinin gene (CGN1) to domestic cow (Bos taurus) syntenic group U29. In turn, a cosmid containing the entire bovine CGN1 was used with fluorescence in situ hybridization to sublocalize this gene to cattle chromosome (Chr) (BTA) 28 band 18. Since BTA 28 and several of the other small acrocentric autosomes of cattle are difficult to discriminate, we have also chromosomally sublocalized CGN1 to the p arm of the lone biarmed autosome of the gaur (Bos gaurus). The use of the gaur 2/28 Robertsonian as a marker chromosome and our assignment of CGN1 to BTA 28 should help resolve some of the nomenclatural questions involving this cattle chromosome.     

Sequence analysis of Vicia faba highly repeated DNA: the BamHI repeated sequence families

Cleavage of Vicia faba nuclear DNA with the restriction endonuclease BamHI yielded discrete size classes of 250, 850, 900, 990, 1 150, 1 500 and 1 750 bp of highly repetitive DNA. Each of these sequence families comprised about 3% of the total genomic DNA. Some sequence members from each sequence family were cloned in pBR322 and their primary structures determined. Computer analyses of nucleotide sequences suggested the existence of about 60 bp sequence periodicity within the repeating unit of the 990 bp sequence family, though the extent of homology among the surmised shorter subrepeat units was very low. With other BamHI sequence families, however, the data did not show any clear internal sequence periodicity. The repeat units of the 850 bp and 1 750 bp sequence families contained nucleotide sequences homologous to the 250 bp family sequence. No sequence relationship between or among other sequence families was observed. There was 13–25% sequence variation among 6 cloned members of the 250 bp family and probably also among those of other BamHI repeat families. DNA sequences homologous to these V. faba BamHI repeat families were detected in Pisum sativum DNA by Southern blot hybridization. Furthermore, very weak cross-hybridization was observed with plant DNAs from Phaseolus vulgaris, Triticum aestivum, Cucumis sativus and Trillium kamtschaticum.     

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.     

Identification of Cu/Zn superoxide dismutase in cattle and river buffaloes

All air-living organisms produce superoxide dismutase (SOD) and several antioxidant enzymes that limit oxidative stress by detoxifying reactive oxygen species. SOD1 gene has been investigated in Egyptian native cattle and buffalos at the level of genomic DNA and cDNA that were extracted from leucocytes. An unexpected band at approximately 370 bp was obtained in cattle genomic DNA and cDNA as well as in buffalo cDNA. SOD1 amplified sequence of native cattle genomic DNA and cDNA showed a 93% alignment. Native cattle genomic DNA SOD1 amplicon shares sequence homology with mRNAs of Bos taurus “similar to superoxide dismutase” (SOD1) sequence of the GeneBank database. It also shares sequence homology with “similar to superoxide dismutase” on B. taurus chromosome BTA13. The results indicate that the genomic DNA of Egyptian native cattle contains SOD1 processed pseudo gene. SOD1 primers amplified three fragments in buffalo genomic DNA which indicates that buffalo genome has different copies of SOD1 due to alternative splicing. It failed to produce the 370 bp fragments found in cattle DNA. The protein analysis revealed no differences between Egyptian native cattle and B. taurus SOD1 mRNA. However, one amino acid, aspartic acid (Asp), in Egyptian native cattle and B. taurus SOD1, is substituted with asparagine (Asn) (D26N) in buffaloes. This amino acid substitution may be due to non-synonymous single nucleotide polymorphisms (nsSNPs). The nsSNPs detected in buffaloes may affect the function of the encoded protein. This study is the first investigation reporting that the resistance of the buffalo to diseases and parasites that afflict cattle may not be acquired but may have a genetic basis.     

Two new polymorphisms in the human elastin gene (ELN)

Two polymorphisms in the human elastin gene have been detected, one with RsaI and a second with BamHI.     

Polymorphism of mitochondrial DNA (mtDNA) in cattle and buffaloes

Mitochondrial DNA (mtDNA) from two breeds of cattle, viz., [Hariana (Bos indicus), Holstein (Bos taurus)] and Indian water buffalo (Bubalis bubalus), was analyzed using 13 restriction endonucleases which recognized an average of about 40 six-base sites. Polymorphism among cattle was detected with six of these enzymes. The two Holstein differed at six sites, whereas the Hariana breed (Bos indicus) did not show any site polymorphism. Surprisingly, the Hariana type differed by only one site from one of the Holstein types. The total size of buffalo mtDNA was estimated to be 16.4 kb. Polymorphism within the Murrah buffalo breed was observed with respect to aBglI site. Scarcely any of the restriction fragments of buffalo mtDNA matched those of cattle mtDNA.     

Identification of a genomic region that complements a temperature-sensitive, high CO2-requiring mutant of the cyanobacterium, Synechococcus sp. PCC7942

Summary In a temperature-sensitive, high CO₂-requiring mutant of Synechococcus sp. PCC7942, the ability to fix intracellularly accumulated inorganic carbon was severely impaired at non-permissive temperature (41° C). In contrast, inorganic carbon uptake and ribulose-1,5-bisphosphate carboxylase activity in the mutant were comparable to the respective values obtained with the wild-type strain. The mutant was transformed to the wild-type phenotype (ability to form colonies at non-permissive temperature under ordinary air) with the genomic DNA of the wild-type strain. A clone containing a 36 kb genomic DNA fragment of the wild-type strain complemented the mutant phenotype. The complementing activity region was associated with internal 17 kb SmaI, 15 kb HindIII, 3.8 kb BamHI and 0.87 kb Pstl fragments. These 4 fragments overlapped only in a 0.4 kb HindIII-PstI region. In the transformants obtained with total genomic DNA or a plasmid containing the 3.8 kb BamHI fragment, the ability to fix intracellular inorganic carbon was restored. Southern hybridization and partial nucleotide sequence analysis indicated that the cloned genomic region was located approximately 20 kb downstream from the structural genes for subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase. The cloned region was transcribed into a 0.5 kb mRNA. These results indicate that the cloned genomic region of Synechococcus sp. PCC7942 is involved in the efficient utilization of intracellular inorganic carbon for photosynthesis.     

Difference Between Xanthomonas campestris pv. phlei and X.c pv. graminis Revealed by Genomic Fingerprinting and Restriction Fragment Length Polymorphism Patterns

Genomic DNA was prepared from 16 strains of Xanthomonas campestris pv. graminis and Xanthomonas campestris pv. phlei isolated from six species of forage grasses in four countries. The two pathovars could be distinguished clearly by genomic fingerprints generated by EcoRI, BamHI or HindIII digestion. DNA profiles produced by HindIII digestion could differentiate not only between the two pathoars but also among strains within the same pahtovar from different countries. A 1.6 kb EcoRI fragment was cloned from genomic DNA of strain LMG726 and used to detect restriction fragment-length polymorphism among the same strains. EcoRI and BamHI polymorphisms were seen between the two pathovars probed with this 1.6 kb EcoRI fragment (p726EI probe). These polymorphisms appeared to be highly conserved and unique for each pathovar, consistent with previous grouping of the strains based on other criteria.     

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.     

Cloning and restriction mapping of the yeast URA2 gene coding for the carbamyl phosphate synthetase aspartate-transcarbamylase complex

Summary Two yeast DNA pools inserted in an hybridEscherichia coli-yeast vector pFL1 were used to transformE. coli and yeast aspartate-transcarbamylase-less strains to prototrophy. From the first pool — aBamHI yeast DNA digest — a 6.4 kbBamHI fragment was recovered that gave good complementation of theE. coli auxotrophy but poor complementation of the yeast auxotrophy. From the second pool — a partialSau3A yeast DNA digest — five independent plasmids complementing eitherE. coli, yeast, or both were recovered. Each of the five plasmids possessed sequences in common with the 6.4 kbBamHI fragment. One of these plasmids, which complemented the twoURA2 activities in yeast and which produced a carbamyl-phosphate synthetase, aspartate-transcarbamylase complex sensitive to UTP feedback inhibition contained the fullURA2 gene. A restriction map of theURA2 gene has been constructed and seven different consecutive segments have been recloned in pBR322 to measure their hybridization withURA2 messenger RNA, allowing us to estimate the limits of the gene.     

DNA polymorphism of the human complementC8β gene: formal genetics and intragenic localization

The eighth component of human complement consists of three subunits of different molecular mass, which are coded for by three separate genetic loci. Polymorphisms have been described at the protein level for the alpha and beta subunits by means of sodium dodecyl sulfate gel electrophoresis and isoelectric focusing. Using a full-length humanC8β cDNA probe, we have studied more than 100 individuals by Southern blot analysis to detect DNA polymorphisms. We have found two restriction fragment length polymorphisms (RFLPs) with the enzymesTaqI andBam HI. TheTaq I polymorphism is defined by two alleles, i.e., a single 4.9 kb fragment or two 2.8/2.1 kb fragments. The allele frequencies are 0.68 and 0.32, respectively. The second RFLP withBam HI is correlated with theTaq I variants: 3 kbBam HI; 4.9 kbTaq I and 3.3 kbBam HI; 2.8/2.1 kbTaqI. Both RFLPs could be mapped to the 3′ portion of theC8β gene. Based on the size of genomic restriction fragments, theC8β gene can be estimated to have a size of 32–36 kb. Because of the even frequency distribution, theC8β DNA polymorphisms may be useful in gene mapping and disease association studies.     

Cloning of DNA sequences from Methanococcus vannielii capable of autonomous replication in yeast

Total DNA of the archaebacterium Methanococcus vannielii was digested with BamHI or BamHI/HindIII, cloned with plasmid Yip5 and analyzed for sequences capable of autonomous replication (ARSs) in the eukaryote Saccharomyces cerevisiae. Two recombinant plasmids were isolated which contained 3.3 kb and 8 kb fragments of methanogen derived DNA with ARS activity. They exhibited low transformation efficiencies for yeast and promoted slow growth of yeast transformants.Abbreviations Ap ampicillin - ARS autonomously replicating sequence - EtBr ethidium bromide - kb kilobase(s) - Mc. Methanococcus - R resistance - RE replication enhancer - RS replication sequence - Tc tetracycline     

β-Lactamase genes of Streptomyces badius,Streptomyces cacaoi and Streptomyces fradiae: Cloning and expression in Streptomyces lividans

Genes encoding extracellular β-lactamases (EC 3.5.2.6) of Gram-positive Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae have been cloned into Streptomyces lividans. The β-lactamase gene of S. badius was initially isolated on a 7 kb BamHI fragment and further located on a 1300 bp DNA segment. An 11 kb BamHI fragment was isolated encompassing the S. cacaoi β-lactamase gene, which was subcloned to a 1250 bp DNA fragment. The β-lactamase gene of S. fradiae was cloned on an 8 kb BamHI fragment and mapped to a 4 kb DNA segment. Each of the three BamHI fragments encompassing the β-lactamase genes hybridized to a BamHI fragment of the corresponding size in chromosomal DNA from the respective strain used for cloning. The activities of the three β-lactamases were predominantly found to be extracellular in the S. lividans recombinants. The S. badius and S. cacaoi β-lactamases exhibited a 10–100-times lower activity in S. lividans, whereas the S. fradiae β-lactamase showed an approximately 10-fold higher activity in the cloned state, compared with the activities found in the original strains.     

Ribosomal DNA variation in foxtail millet, Setaria italica (L.) P. Beauv., and a survey of variation from Europe and Asia

 Restriction fragment length polymorphism (RFLP) and the structure of ribosomal RNA genes (rDNA) were investigated in 117 landraces of foxtail millet, Setaria italica (L.) P. Beauv. Five RFLP phenotypes were found when the genomic DNA was digested with BamHI; these were named types I–V. Of these types I, II and III were the most frequent. Type I was mainly distributed in the temperature zone, type II in the Taiwan-Philippines Islands and type III in South Asia. Restriction mapping of the cloned rDNA and comparison with RFLP phenotypes showed that the different types originated from a polymorphism in the length within the intergenic spacer (IGS) and BamHI site changes within the IGS. Received: 28 August 1996 / Accepted: 28 February 1997     

Isolation of a new restriction enzyme,ApaCI, an isoschizomer ofBamHI produced byAcetobacter pasteurianus

A new Type II restriction endonucleaseApaCI purified fromAcetobacter pasteurianus is an isoschizomer ofBamHI that cleaves at the nucleotide sequence 5′-G/GATCC-3′ of double-stranded DNA. The single restriction activity present in this strain permits rapidly purified 30 000 units of cleavage activity from 10 g of freshly harvested cells. The resultingApaCI preparation is free of contaminant nuclease activities that might interfere within vitro manipulation of DNA.     

Microcloning of maize chromosome 9 by using a flow-sorting technique

We constructed a chromosome 9 lambda DNA library from flow-sorted maize chromosomes. Approximately 3 million maize chromosome 9 were collected with high purity by flow cytometric sorting of chromosomes isolated from an oat-maize chromosome 9 addition line based on the cytogram of fluorescent pulse area versus fluorescent pulse width. Chromosome 9 DNA was partially digested withBamH I, dephosphorylated, and ligated with arms ofBamH I-digested lambda DASH vector (Stratagene). A total of 2.0×10⁶ independent recombinants with an average insert size of 15 kb were obtained. For a 99% probability that every sequence of chromosome 9 is represented in at least one chimeric phage, 5.6×10⁴ cloned fragments are needed. This library covers the entire maize chromosome 9. Hybridizing cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. This individual chromosome library is useful in plant genome mapping and gene isolation.     

Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis

Summary Cloning in Escherichia coli and Bacillus subtilis was carried out using the bifunctional plasmid pDH5060. B. subtilis chromosomal DNA and pDH5060 DNA were digested with either BamHI or SalI, then annealed, ligated, and transformed into E. coli SK2267. Transformants containing sequences ligated into the BamHI or SalI sites in the Tc^r gene of pDH5060 were selected directly using a modification of the fusaric acid technique. The BamHI and SalI clone banks contain about 250 and 140 B. subtilis fragments, respectively, with an average insert size of 8–9 Kbp in the BamHI and 4–5 Kbp in the SalI bank. The inserts ranged in size from 0.3 Kbp to greater than 20 Kbp. The vector used here therefore accepts inserts which are significantly larger than previously reported for other B. subtilis cloning systems. All individual cloned B. subtilis sequences examined were stably propagated in E. coli SK2267. Eight of eighteen B. subtilis auxotrophic markers tested (aroG, gltA, glyB, ilvA, metC, purA, pyrD, and thrA) were transformed to prototrophy with BamHI or SalI clone bank DNA. All or part of the hybrid plasmid DNA recombined at the sites of homology in the chromosome of these Rec⁺ recipients. Loss of sequences from hybrid plasmids was not prevented in a r ^- m ^- recE4 recipient strain of B. subtilis. Although the recE4 background prevented recombination between homologous chromosomal DNA, a variety of cloned fragments were shown to be unstable and undergo deletions of both insert and plasmid sequences. In addition, B. subtilis sequences propagated in E. coli transformed B. subtilis recE4 recipients with a 500-1,000-fold reduced efficiency.     

Localization of chromosome breakpoints induced by DNase I in Chinese hamster ovary (CHO) cells

DNase I was electroporated into S-phase CHO cells and induced chromosome breakpoints were localized in G-banded metaphases. More than 75% of breakpoints mapped to Giemsa-light bands, 18% to Giemsa-dark bands and about 7% to band junctions. Chromosome breakpoint clusters produced by DNase I colocalized with chromosome breakpoints induced by the restriction endonucleases AluI and BamHI in the G1- and S-phases of the cell cycle in CHO cells. Digestion of metaphase spreads with AluI, BamHI and DNase I produced G-bands, indicating that G-light bands are more sensitive to endonuclease action. The possible role of nuclease-sensitive sites in active chromatin as selective targets for the induction of chromosome breakpoints by these endonucleases is discussed. Received: 15 January 1997; in revised form: 21 July 1997 / Accepted: 23 July 1997