Restriction fragment length polymorphisms in the D7S1 region of human chromosome 7

Summary A restriction fragment length polymorphism in the D7S1 region of chromosome 7 is detected by hybridizing the recombinant plasmid pA2H3 to HindIII- or HinfI-digested human DNA. Three HindIII and two HinfI alleles were detected, and it was found that all individuals carrying the variant HinfI allele also had the commoner of the two variant HindIII alleles. All variants seem to result from point mutation in restriction enzyme recognition sites. Restriction mapping of the D7S1 region revealed that the associated HindIII and HinfI alleles are defined by sites 300 base-pairs (bp) apart, and it is suggested that the close proximity of these sites is sufficient to account for the strong phenotype association observed.     

Integration of eukaryotic genes for 5S RNA and histone proteins into a phage lambda receptor.

Highly purified HindIII restriction fragments of Xenopus laevis 5S DNA and of Psammechinus miliaris histone DNA have been covalently inserted into a derivative of phage lambda. This phage, genetically constructed by Murray et al. (1), contains only a single target for HindIII in the cI gene. Viable hybrid molecules were detected as clear plaque-forming phage after transfection of E. coli, the vast majority of which were shown by hybridization to be recombinants of the desired type. The lambdaSam7 mutation has been introduced into one hybrid phage containing histone DNA, thereby substantially increasing the yield of recombinant DNA.     

麦迪霉素产生菌具有启动功能的DNA片段的克隆和分析

利用启动子探针质粒载体pIJ486从麦迪霉素产生菌总DNA中克隆得到了一段具有启动功能的DNA片段.通过限制性酶酶切分析,测定插入DNA片段大小为2.3kb.又利用载体pIJ486和pIJ487的新霉素抗性结构基因上游有多酶切点方向相反的性质,分析了插入片段在两个不同方向上的启动能力.结果表明,在两个方向上均有启动功能,但强弱相差六倍.其中在XbaI-HindIII方向上具有较强的启动能力,在变铅青链霉菌中新霉素抗性水平可达20mg/ml以上.进一步对插入片段的三个BamHI小片段进行分析的结果表明,较强启动子区域集中在BamHI-BamHI 0.79kb DNA片段上.     

Cloning and characterization of genes coding for ribosomal RNA inCampylobacter jejuni

The DNA fragments coding for ribosomal RNA inCampylobacter jejuni have been cloned from a genomic library ofC. jejuni constructed inEscherichia coli. Clones carrying DNA Sequences for rRNA were identified by hybridization of 5-end-labeled rRNA fromC. jejuni to colony blots of transformants from this gene library. Cloned DNA sequences homologous to each of 5S, 16S, and 23S rRNA were idenfified by hybridization of labeled plasmid DNA to Northern blots of rRNA. The gene coding for 23S rRNA was found to be located on a 5.5kb HindIII fragment, while the 5S and 16S rRNA genes were on HindIII fragments of 1.65 and 1.7 kb, respecitively. The DNA fragment containing the 16S rRNA gene was characterized by restriction endonuclease mapping, and the location of the 16S rRNA gene on this fragment was determined by hybridization of 5-end-labeled rRNA to restriction fragments and also by DNA sequence determination. It appears that the major portion of the coding region for 16S rRNA is located on the 1.7-kb HindIII fragment, while a small portion is carried on an adjacent HindIII fragment of 7.5 kb. Cloned rRNA genes fromC. jejuni were used to study the organization of the rDNA inC. jejuni and other members of the genùsCampylobacter.     

The organization of ribosomal RNA genes in the mitochondrial DNA of Tetrahymena pyriformis strain ST.

1. We have constructed a physical map of the mtDNA of Tetrahymena pyriformis strain ST using the restriction endonucleases EcoRI, PstI, SacI, HindIII and HhaI. 2. Hybridization of mitochondrial 21 S and 14 S ribosomal RNA to restriction fragments of strain ST mtDNA shows that this DNA contains two 21-S and only one 14-S ribosomal RNA genes. By S1 nuclease treatment of briefly renatured single-stranded DNA the terminal duplication-inversion previously detected in this DNA (Arnberg et al. (1975) Biochim. Biophys. Acta 383, 359--369) has been isolated and shown to contain both 21-S ribosomal RNA genes. 14 S ribosomal RNA hybridizes to a region in the central part of the DNA, about 8000 nucleotides or 20% of the total DNA length apart from the nearest 21 S ribosomal RNA gene. 3. We have confirmed this position of the three ribosomal RNA genes by electron microscopical analysis of DNA . RNA hybrid molecules and R-loop molecules. 4. Hybridization of 21 S ribosomal RNA with duplex mtDNA digested either with phage lambda-induced exonuclease or exonuclease III of Escherichia coli, shows that the 21-S ribosomal RNA genes are located on the 5'-ends of each DNA strand. Electron microscopy of denaturated mtDNA hybridized with a mixture of 14-S and 21-S ribosomal RNAs show that the 14 S ribosomal RNA gene has the same polarity as the nearest 21 S ribosomal RNA gene. 5. Tetrahymena mtDNA is (after Saccharomyces mtDNA) the second mtDNA in which the two ribosomal RNA cistrons are far apart and the first mtDNA in which one of the ribosomal RNA cistrons is duplicated.     

DNA polymorphism in the Mongolian population. Restriction fragment length polymorphism analysis of DNA at seven loci of the nuclear genome]

Seven DNA markers from five genes and one chromosomal region were analysed in Mongolian population using the polymerase chain reaction. The frequencies of alleles of the polymorphisms detected with HindIII in the HBG-2, AvaII in the HBB, MspI and XbaI in the Apo-B, PstI in the D7S8, HincII in the LDLR and allele frequency of the minisatellite fragment in the AT-3 have been determined. The results of the RELP for Apo-B(MspI), LDLR, D7S8 and AT-3 are obtained for the first time among Mongoloids. DNA markers studied demonstrated high level of polymorphisms in the population of Mongolia, except for XbaI and MspI restriction sites at the Apo-B locus. The data obtained for Mongolian population and the literature data were compared.     

Cloning and mapping of infA, the gene for protein synthesis initiation factor IF1.

The gene for translation initiation factor IF1, infA, has been identified by using two synthetic oligonucleotides to screen a Charon 30 library of Escherichia coli DNA. A recombinant lambda phage, C1921, was purified from a plaque positive for both probes. A 2.8 kb BglII fragment and a 2.0 kb HindIII fragment isolated from C1921 were subcloned into the BamHI and HindIII sites of pBR322 to yield pTB7 and pTH2 respectively. Synthesis of IF1 in maxicells transformed with pTB7 or pTH2 indicates the presence of inf A in both inserts. This was confirmed by DNA sequencing: a region was found that codes for a 8,119 dalton protein with an amino acid sequence corresponding to IF1. The chromosomal location of inf A was determined by mapping the closely linked beta-lactamase gene (Ampr) in pTB7 and pTH2. pTB7 and pTH2 were transformed into polA Hfr hosts, and integration of the plasmid by homologous recombination near inf A was selected on the basis of ampicillin resistance. The site of integration was confirmed by Southern blot analysis of restriction nuclease digested wild type and transformed genomic DNA. The Ampr marker (and therefore inf A) was mapped to about 20 minutes by Hfr interrupted matings and P1 transduction experiments. The structure and regulation of the inf A operon currently are being investigated.     

Hybridizable sequences between cytoplasmic ribosomal RNAs and 3 micron circular DNAs of Saccharomyces cerevisiae and Torulopsis glabrata.

We have shown that 2.8 and 3.1 micron circular DNA molecules, previously reported to be present in Saccharomyces cerevisiae and Torulopsis glabrata respectively, contain sequences hybridizing to cytoplasmic ribosomal RNAs. In S. cerevisiae the 2.8 micron circular DNA appears to be identical to the rDNA repeating unit from nuclear DNA, both in length (approximately 9000 base pairs) and in the location of the 25, 18 and 5.8S rRNA sequences on the large HindIII fragment (6500 bp) and the presence of the 5S rRNA sequence on the small HindIII fragment. The 3.1 micron molecule from T. glabrata is approximately 2000 base pairs longer than the S. cerevisiae molecule and in addition, one of the HindIII sites lies within the region hybridizing to 25, 18 and 5.8S rRNAs. In S. cerevisiae the 4-5 copies of the 2.8 micron circular DNA molecules per cell, which have an extra-nuclear location, do not appear to be essential for cell viability as in one strain they were undetectable.     

Cloning and characterization of bacteriophage-like DNA from Haemophilus somnus homologous to phages P2 and HP1.

In an attempt to identify and characterize components of a heme uptake system of Haemophilus somnus, an Escherichia coli cosmid library of H. somnus genomic DNA was screened for the ability to bind hemin (Hmb+). The Hmb+ phenotype was associated with a 7,814-bp HindIII fragment of H. somnus DNA that was subcloned and sequenced. Thirteen open reading frames (orfs) were identified, all transcribed in one direction, and transposon mutagenesis identified orf7 as the gene associated with the Hmb+ phenotype. Orf7 (178 amino acids) has extensive homology with the lysozymes of bacteriophages P-A2, P21, P22, PZA, phi-29, phi-vML3, T4, or HP1. The orf7 gene complemented the lytic function of the K gene of phage P2 and the R gene of phage lambda. A lysozyme assay using supernatants from whole-cell lysates of E. coli cultures harboring plasmid pRAP501 or pGCH2 (both of which express the orf7 gene product) exhibited significant levels of lysozyme activity. The orf6 gene upstream of orf7 has the dual start motif common to the holins encoded by lambdoid S genes, and the orf6 gene product has significant homology to the holins of phages HP1 and P21. When expressed from a tac promoter, the orf6 gene product caused immediate cell death without lysis, while cultures expressing the orf7 gene product grew at normal rates but lysed immediately after the addition of chloroform. Based on this data, we concluded that the Hmb+ phenotype was an artifact resulting from the expression of cloned lysis genes which were detrimental to the E. coli host. The DNA flanking the cloned lysis genes contains orfs that are similar to structural and DNA packaging genes of phage P2. Polyclonal antiserum against Orf2, which is homologous to the major capsid precursor protein (gpN) of phage P2, detected a 40,000-M(r) protein expressed from pRAP401 but did not detect Orf2 in H. somnus, lysates. The phage-like DNA was detected in the serum-susceptible preputial strains HS-124P and HS-127P but was absent from the serum-resistant preputial strains HS-20P and HS-22P. Elucidation of a potential role for this cryptic prophage in the H. somnus life cycle requires more study.     

Beta-globin gene haplotypes in Polynesians are predominantly southern Chinese in type

beta-Globin gene haplotypes obtained in Polynesian Samoans were similar to those described in Southern Chinese. An atypical HindIII restriction fragment length polymorphism detected with pRK29, a 3' beta-globin gene probe, was present at a gene frequency of 7% in Samoans. Haplotype patterns suggest that this polymorphism may have arisen by 1 or 2 mutational events. DNA haplotypes derived from the beta-globin gene cluster confirm nuclear and mitochondrial DNA data that Polynesian precursor populations were East Asian in origin.     

Organization of the yeast ribosomal RNA gene cluster via cloning and restriction analysis.

The restriction endonuclease EcoR1 cleaves Saccharomyces cerevisiae DNA, which codes for ribosomal RNA (rRNA), into seven fragments, A second restriction endonuclease, HindIII, cleaves the same yeast ribosomal DNA into two fragments. These two restriction enzymes each yield DNA segments that total about 5.9 megadaltons. The "repeat unit" of the yeast genes coding for rRNA is thus about 5.9 megadaltons or about 9000 base pairs long. The two HindIII-cleaved DNA fragments as well as one of the EcoR1-cleaved DNA fragments were purified and amplified by cloning in Escherichia coli. Three of the seven EcoR1-generated DNA fragments could then be ordered by treating the two cloned HindIII DNA fragments with EcoR1. This led the assignment of the two HindIII restriction sites. The various restriction DNA fragments were hybridized directly from the gel utilizing 32P-labeled 5 S, 5.8 S, 18 S, and 25 S rRNA. Identification of the various DNA restriction segments then led to the final ordering of the DNA fragments. The gene coding for the 5 S RNA is adjacent to the gene coding for the 35 S precursor rRNA. These two groups of genes thus occur as a cluster in the following sequence: [5 S-spacer]-[spacer-18 S-5.8 S-25 S-spacer]-[spacer-5 S]. The actual map of the DNA restriction fragments is presented.     

Characterization of avian myeloblastosis-associated virus DNA intermediates.

The major species of unintegrated linear viral DNA identified in chicken embryonic fibroblasts infected with either the avian myeloblastosis-associated viruses (MAV-1, MAV-2) or the standard avian myeloblastosis virus complex (AMV-S) has a mass of 5.3 X 10(6) daltons. An additional minor DNA component observed only in AMV-S-infected cells has a mass of 4.9 X 10(6) daltons. The unintegrated linear viral DNAs and integrated proviruses of MAV-1 and MAV-2 have been analyzed by digestion with the restriction endonucleases EcoRI and HindIII. MAV-2 lacks a HindIII site present in MAV-1. These fragments have been compared to those generated by EcoRI and HindIII digestion of linear viral DNAs of AMV-S. Restriction enzyme digestion of AMV-S viral DNA produced unique fragments not found with either MAV-1 or MAV-2 viral DNAs. The major viral component present in AMV-S stocks has the HindIII restriction pattern of MAV-1. Restriction enzyme analysis of the 5.3 X 10(6)-dalton unintegrated MAV viral DNAs and their integrated proviruses suggests that the DNAs have a direct terminal redundancy of approximately 0.3 megadaltons and integrate colinearly with respect to the unintegrated linear DNA.     

Evaluation of a ribosomal RNA gene probe for the identification of species and subspecies within the genus Staphylococcus.

To evaluate a 16S rRNA gene probe for the identification of staphylococcal species and subspecies, we have augmented previous studies involving 12 staphylococcal species by analysing the remaining 16 species currently classified in the genus Staphylococcus. HindIII- and EcoRI-restricted DNA of isolates from validly described species of Staphylococcus was probed with radiolabelled plasmid pBA2 containing 16S rDNA from Bacillus subtilis. The Dice coefficient was used to assess similarity between the 74 HindIII- and the 81 EcoRI-hybridization patterns obtained from a total of 271 isolates belonging to 31 staphylococcal taxa (28 species, of which three include two subspecies). The use of HindIII yielded a better discrimination of the staphylococci than the use of EcoRI. All of the isolates belonging to the same species or subspecies, except S. hyicus isolates, were recovered as homogeneous clusters using their HindIII hybridization patterns. The phenotypically close taxa were clearly distinguished. Thus, the method presented in this study constitutes a powerful tool for the identification of taxa within the genus Staphylococcus.     

苜蓿中华根瘤菌与耐盐有关DNA片段的亚克隆和测序分析

将苜蓿中华根瘤菌(Sinorhizobium meliloti)042B与耐盐有关的4kb ClaⅠ DNA片段克隆在pML122上,用HindⅢ酶切下其2.4kb DNA片段,回收后与pBBR1\|MCS2连接,然后转化大肠杆菌(Escherichia coli)DH5α,筛选到转化子GS2〖DK〗。将残留在pML122上16kb ClaⅠ\|HindⅢ DNA片段连同质粒一起回收,让其自连,转化大肠杆菌S17-1,得到转化子GS0。以GS0为供体,042B的盐敏感突变株GZ17为受体,进行二亲本杂交,没有得到接合子。以GS2为供体,GZ17为受体,在辅助质粒pRK2013的协助下,进行三亲本杂交,筛选到接合子GG2,获得2.4kb HindⅢ与耐盐有关的DNA片段。将此片段连接到测序载体pGEM\|7Zf(+)上进行测序。测序结果表明,该24kb HindⅢ DNA片段含有3个开放阅读框(ORF)。在此基础上再一次亚克隆,获得19kb与耐盐有关的DNA片段。     

Nucleosome arrangement in green monkey alpha-satellite chromatin. Superimposition of non-random and apparently random patterns

We have studied the structure of tandemly repetitive alpha-satellite chromatin (alpha-chromatin) in African green monkey cells (CV-1 line), using restriction endonucleases and staphylococcal nuclease as probes. While more than 80% of the 172-base-pair (bp) alpha-DNA repeats have a HindIII site, less than 15% of the alpha-DNA repeats have an EcoRI site, and most of the latter alpha-repeats are highly clustered within the CV-1 genome. EcoRI and HindIII solubilize approximately 8% and 2% of the alpha-chromatin, respectively, under the conditions used. EcoRI is thus approximately 30 times more effective than HindIII in solubilizing alpha-chromatin, with relation to the respective cutting frequencies of HindIII and EcoRI on alpha-DNA. EcoRI and HindIII solubilize largely non-overlapping subsets of alpha-chromatin. The DNA size distributions of both EcoRI- and HindIII-solubilized alpha-chromatin particles peak at alpha-monomers. These DNA size distributions are established early in digestion and remain strikingly constant throughout the digestion with either EcoRI or HindIII. Approximately one in every four of both EcoRI- and HindIII-solubilized alpha-chromatin particles is an alpha-monomer. Two-dimensional (deoxyribonucleoprotein leads to DNA) electrophoretic analysis of the EcoRI-solubilized, sucrose gradient-fractionated alpha-oligonucleosomes shows that they do not contain "hidden" EcoRI cuts. Moreover, although the EcoRI-solubilized alpha-oligonucleosomes contain one EcoRI site in every 172-bp alpha-DNA repeat, they are completely resistant to redigestion with EcoRI. This striking difference between the EcoRI-accessible EcoRI sites flanking an EcoRI-solubilized alpha-oligonucleosome and completely EcoRI-resistant internal EcoRI sites in the same alpha-oligonucleosome indicates either that the flanking EcoRI sites occur within a modified chromatin structure or that an altered nucleosome arrangement in the vicinity of a flanking EcoRI site is responsible for its location in the nuclease-sensitive internucleosomal (linker) region. Analogous redigestions of the EcoRI-solubilized alpha-oligonucleosomes with either HindIII, MboII or HaeIII (both before and after selective removal of histone H1 by an exchange onto tRNA) produce a self-consistent pattern of restriction site accessibilities. Taken together, these data strongly suggest a preferred nucleosome arrangement within the EcoRI-solubilized subset of alpha-oligonucleosomes, with the centers of most of the nucleosomal cores being approximately 20 bp and approximately 50 bp away from the nearest EcoRI and HindIII sites, respectively, within the 172-bp alpha-DNA repeat. However, as noted above, the clearly preferred pattern of nucleosome arrangement within the EcoRI-solubilized alpha-oligonucleosomes is invariably violated at the ends of every such alpha-oligonucleosomal particle, suggesting at least a partially statistical origin of this apparently non-random nucleosome arrangement.(ABSTRACT TRUNCATED AT 400 WORDS)     

Physical map of the origin of defective DNA in herpes simplex virus type 1 DNA.

The origin of defective DNA (dDNA) of the Patton strain of herpes simplex virus type 1 (HSV-1) was physically mapped with BamHI in the parental DNA. The dDNA obtained from virus passaged at high multiplicities of infection was resistant to cleavage with HindIII, whereas digestion with EcoRI yielded a cluster of fragments 5.4 to 5.7 megadaltons (Mdal) in size. Cleavage with BamHI gave a cluster of fragments 2.6 to 3.2 Mdal in size, plus two homogeneous, comigrating 1-Mdal fragments. One of the latter fragments contained the single EcoRI site approximately 65 base pairs from one end. Hybridization of in vitro labeled dDNA probe to EcoRI, HindIII, BamHI, and Hpa I digests of nondefective HSV-1 DNA demonstrated that, in addition to the S-region terminal repeat, only one end of the S region was involved in the generation of this class of dDNA. Thus, the dDNA probe did not hybridize to either the S region 3.0-Mdal HindIIIN fragment or a 3.0-Mdal BamHI fragment of the adjacent 8.7-Mdal HindIIIG fragment, but did hybridize to four BamHI fragments of HindIII G (approximately 5.7 Mdal). The cluster of 2.6- to 3.2-Mdal fragments obtained with BamHI digestion of dDNA appears to represent a novel junction between the termination of dDNA adjacent to the 3.0-Mdal BamHI fragment in HindIII G and the 2.0- to 2.3-Mdal BamHI fragment terminal in HSV-1 DNA.     

Mutational analyses of restriction endonuclease-HindIII mutant E86K with higher activity and altered specificity

We have performed mutational analyses of restriction endonuclease HindIII in order to identify the amino acid residues responsible for enzyme activity. Four of the seven HindIII mutants, which had His-tag sequences at the N-termini, were expressed in Escherichia coli, and purified to homogeneity. The His-tag sequence did not affect enzyme activity, whereas it hindered binding of the DNA probe in gel retardation assays. A mutant E86K in which Lys was substituted for Glu at residue 86 exhibited high endonuclease activity. Gel retardation assays showed high affinity of this mutant to the DNA probe. Surprisingly, in the presence of a transition metal, Mo(2+) or Mn(2+), the E86K mutant cleaved substrate DNA at a site other than HindIII. Substitution of Glu for Val at residue 106 (V106E), and Asn for Lys at residue 125 (K125N) resulted in a decrease in both endonucleolytic and DNA binding activities of the enzyme. Furthermore, substitution of Leu for Asp at residue 108 (D108L) abolished both HindIII endonuclease and DNA binding activities. CD spectra of the wild type and the two mutants, E86K and D108L, were similar to each other, suggesting that there was little change in conformation as a result of the mutations. These results account for the notion that Asp108 could be directly involved in HindIII catalytic function, and that the substitution at residue 86 may bring about new interactions between DNA and cations.