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PubMed1988

DNA divergence in and around the alcohol dehydrogenase locus in five closely related species of Hawaiian Drosophila.

The alcohol dehydrogenase (Adh) region from five planitibia subgroup species of Hawaiian picture-wing Drosophila has been cloned. A total of 15 kb of DNA in and around the Adh gene has been compared among the five species. Genetic distances were calculated to determine evolutionary relationships. These distances agree with previous distances determined by protein polymorphism and DNA hybridization techniques and can be interpreted in terms of specific island colonization and speciation (founder) events over the past 5 Myr. Examination of the restriction maps of the cloned Adh region from the five species shows many instances of small deletions, insertion of a transposable element in D. heteroneura, and the existence of a highly variable region on the 3' side of the Adh gene. Clustering relationships and rates of DNA change are calculated and compared with the relationship found for other species of Drosophila.

Alcohol Dehydrogenase↗
PubMed1988

Construction of an Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp.

A plasmid transformation system for Rhodococcus sp. strain H13-A was developed by using an Escherichia coli-Rhodococcus shuttle plasmid constructed in this study. Rhodococcus sp. strain H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200, and pMVS300, of 75, 19.5, and 13.4 kilobases (kb), respectively. A 3.8-kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3-kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla), as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1-kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1(pMVS301) and transformed into Rhodococcus sp. strain AS-50, a derivative of strain H13-A, by polyethylene glycol-assisted transformation of Rhodococcus protoplasts and selection for thiostrepton-resistant transformants. Thiostrepton-resistant transformants were also ampicillin resistant and were shown to contain pMVS301, which was subsequently isolated and transformed back into E. coli. The cloned 3.8-kb fragment of Rhodococcus DNA in pMVS301 contains a Rhodococcus origin of replication, since the hybrid plasmid was capable of replication in both genera. The plasmid was identical in E. coli and Rhodococcus transformants as determined by restriction analysis and was maintained as a stable, independent replicon in both organisms. Optimization of the transformation procedure resulted in transformation frequencies in the range of 10(5) transformants per micrograms of pMVS301 DNA in Rhodococcus sp. strain H13-A and derivative strains. The plasmid host range extends to strains of Rhodococcus erythropolis, R. globulerus, and R. equi, whereas stable transformants were not obtained with R. rhodochrous or with several coryneform bacteria tested as recipients. A restriction map demonstrated 14 unique restriction sites in pMVS301, some of which are potentially useful for molecular cloning in Rhodococcus spp. and other actinomycetes. This is the first report of plasmid transformation and of heterologous gene expression in a Rhodococcus sp.

Ampicillin↗
PubMed1991

Detection of elastase production in Escherichia coli with the elastase structural gene from several non-elastase-producing strains of Pseudomonas aeruginosa.

The elastase structural gene from Pseudomonas aeruginosa IFO 3455 has been cloned and sequenced. Using this gene as a probe, we cloned the DNA fragments (pEL3080R, pEL10, and pEL103R) of the elastase gene from non-elastase-producing strains (P. aeruginosa IFO 3080, N-10, and PA103 respectively). These three Pseudomonas strains showed no detectable levels of elastase antigenicity by Western blotting (immunoblotting) or by elastase activity. When elastase structural genes about 8 kb in length were cloned into pUC18, an Escherichia coli expression vector, we were able to detect both elastase antigenicity and elastolytic activity in two bacterial clones (E. coli pEL10 and E. coli pEL103R). However, neither elastolytic activity nor elastase antigenicity was detected in the E. coli pEL3080R clone, although elastase mRNA was observed. The partial restriction map determined with several restriction enzymes of these three structural genes corresponded to that of P. aeruginosa IFO 3455. We sequenced the three DNA segments of the elastase gene from non-elastase-producing strains and compared the sequences with those from the elastase-producing P. aeruginosa strains IFO 3455 and PAO1. In P. aeruginosa N-10 and PA103, the sequences were almost identical to those from elastase-producing strains, except for several nucleotide differences. These minor differences may reflect a microheterogeneity of the elastase gene. These results suggest that two of the non-elastase-producing strains have the normal elastase structural gene and that elastase production is repressed by regulation of this gene expression in P. aeruginosa. Possible reasons for the lack of expression in these two strains are offered in this paper. In P. aeruginosa IFO 3080, the sequence had a 1-base deletion in the coding region, which should have caused a frameshift variation in the amino acid sequence. At present, we have no explanation for the abnormal posttransciptional behavior of this strain.

Amino Acid Sequence↗
PubMed1978

DNA of Epstein-Barr virus. IV. Linkage map of restriction enzyme fragments of the B95-8 and W91 strains of Epstein-Barr Virus.

The arrangement of EcoRI, Hsu I, and Sal I restriction enzyme sites in the DNA of the B95-8 and W91 isolates of Epstein-Barr virus (EBV) has been determined from the size of the single-enzyme-cleaved fragments and from blot hybridizations that identify which fragments cut from the DNA with one enzyme contain nucleotide sequences in common with fragments cut from the DNA with a second enzyme. The DNA of the B95-8 isolate was the prototype for this study. The data indicate that (i) approximately 95 X 10(6) to 100 X 10(6) daltons of EBV (B95-8) DNA is in a consistent and unique sequence arrangement. (ii) Both termini are variable in length. One end of the molecule after Hsu I endonuclease cleavage consists of approximately 3,000 base pairs, with as many as 10 additional 500-base pair segments. The opposite end of the molecule after Sal I endonuclease cleavage consists of approximately 1,500 base pairs, with as many as 10 additional 500-base pair segments. (iii) The opposite ends of the molecule contain homologous sequences. The high degree of homology between the opposite ends of the molecule and the similarity in size of the "additional" 500-base pair segments suggests that there are identical repeating units at both ends of the DNA. The arrangement of restriction endonuclease fragments of the DNA of the W91 isolate of EBV is similar to that of the B95-8 isolate and differs from the latter in the presence of approximately 7 X 10(6) daltons of "extra" DNA at a single site. Thus, the size of almost all EcoRI, Hsu I, and Sal I fragments of EBV (W91) DNA is identical to that of fragments of EBV (B95-8) DNA. A single EcoRI fragment, C, of EBV (W91) DNA is approximately 7 X 10(6) daltons larger than the corresponding EcoRI fragment of EBV (B95-8) DNA. Digestion of EBV (W91) DNA with Hsu I or Sal I restriction endonucleases produces two fragments (Hsu I D1 and D2 or Sal I G2 and G3) which differ in total size by approximately 7 X 10(6) daltons from the fragments of EBV (B95-8) DNA. Furthermore, the EcoRI, Hsu I, and Sal I fragments of EBV (W91) and (B95-8) DNAs, which are of similar molecular weight, have homologous nucleotide sequences. Moreover, the W91 fragments contain only sequences from a single region of the B95-8 genome. Two lines of evidence indicate that the "extra" sequences present in W91 EcoRI fragment C are viral DNA and not cellular. (i) The molecular weight of the "enlarged" EcoRI C fragment of EBV (W91) DNA is identical to that of the EcoRI C fragment of another isolate of EBV (Jijoye), (ii) The HR-1 clone of Jijoye has previously been shown to contain DNA which is not present in the B95-8 strain but is present in the EcoRI C and Hsu I D2 and D1 fragments of EBV (W91) DNA (N. Raab-Traub, R. Pritchett, and E. Kieff, J. Virol. 27:388-398, 1978).

Cell Line↗
PubMed2001

Delineation of a 150-kb breakpoint cluster in benign thyroid tumors with 19q13.4 aberrations.

Structural rearrangements involving the long arm of chromosome 19 characterize a cytogenetic subgroup of benign thyroid tumors and constitute one of the most frequent specific chromosome abnormalities in epithelial tumors. Recently, we have been able to narrow down the breakpoint region affected in two cell lines to a region covered by a single PAC clone. Close to that region a candidate gene has been identified which we tentatively referred to as RITA (Rearranged In Thyroid Adenomas) now named ZNF331 according to HUGO nomenclature. However, the results had been obtained on two cell lines only making it necessary to extend the studies to a larger number of tumors including primary material. Herein, we have used four further primary tumors showing translocations involving 19q13 for fluorescence in situ hybridization (FISH) mapping studies using a variety of molecular probes from a 470-kbp cosmid/BAC contig. Ten new STSs were characterized and physically mapped within an EcoRI restriction map. The results enabled us to define an approximately 150-kbp breakpoint cluster region of the 19q13 aberrations in benign thyroid tumors flanked by two newly established STS markers.

Adenoma↗
PubMed1986

Molecular genetics and the characterization of steroid 21-hydroxylase deficiency.

Classical 21-hydroxylase deficiency congenital adrenal hyperplasia is a monogenic autosomal recessive disorder that has been conclusively shown by family HLA-typing studies to be in close genetic linkage with the human major histocompatibility complex. More recently recognized is the nonclassical disorder, an attenuated form of 21-hydroxylase deficiency characterized variably by late onset or absence of symptoms. Certain of the mild 21-hydroxylase deficiency allotypes involved in the nonclassical disorder have also been shown to be genetically linked with HLA, exhibiting distinct (B and B,DR) antigen associations. The nonclassical disorder is now also known to result from different genotypes: two mild 21-hydroxylase defects in conjunction, or a mild defect occurring with a sever (classical) defect. Restriction mapping and hybridization analysis have located two highly homologous base sequences, one structural gene coding for 21-hydroxylase and one pseudogene, in the Class III region of the MHC in tandem with the A and B genes for C4, the fourth component of complement. Current work documenting and characterizing gene abnormalities, as well as elucidating the molecular genetic basis of the mutations that have arisen, is aimed at developing better cDNA probes for prenatal diagnosis by amniocentesis and chorionic villus biopsy. In addition, because of the close association of the C4 and 21-hydroxylase genes, coordination of data on C4 variants and null alleles with altered 21-hydroxylase activity is improving understanding of the genetic mechanisms generating disease alleles of this enzyme crucial for normal endocrine function.

17-alpha-Hydroxyprogesterone↗
PubMed1997

Physical mapping of two histone gene clusters on human chromosome 6p22.1-22.2.

Histones are basic proteins which are responsible for the assembly and maintenance of the nucleosomal structure within the chromosomal fiber in eukaryotes. Two clusters of these genes have previously been mapped to the region 6p21.1-p22.2. We describe here a radiation hybrid map, a long range restriction map and a YAC contig covering and linking these two clusters and giving the precise localisation with respect to the HLA complex. The large cluster contains five H1 histone genes in the 6p22.2 region, the smaller only one, H1F5 (H1.5), in 6p22.1. In both clusters, each H1 locus is accompanied by several core histone genes. The large cluster has additionally been covered by a sequence ready PAC contig and three probably unrelated genes (TRMI2, BTN and SSADH) have been accurately localized within the 6p22.2-p22.1 region.

Chromosomes, Human, Pair 6↗
PubMed1991

[Studies of mtDNA of Ustilago maydis. I. Cloning and gene mapping].

This paper covers the following studies of mtDNA of Ustilago maydis. (1) By inserting the Bam HI and Pst I fragments of the mtDNA into the corresponding sites of pBR322, we cloned a unique sequence of 49.6 kb, accounting for 89.3% of the mitochondrial genome (60.7 kb). (2) With heterogenous genes from plants or fungi as probes, we identified seven genes, and mapped them onto the restriction map of the mt DNA. The genes were arranged in such an order: -UmCOB-UmOXII-S-rR NA-UmOXIII-L-rRNA-UmATPase6-UmOXI-. (3) We tried to express the three cloned genes, UmOXII, UmOXIII, and Um-ATPase 6, in E. coli maxcel expression system, but no specific protein was observed.

Chromosome Mapping↗
PubMed1987

The deletion in both common types of hereditary persistence of fetal hemoglobin is approximately 105 kilobases.

The most common forms of hereditary persistence of fetal hemoglobin (HPFH) involve large deletions that remove the adult delta and beta genes but leave the paired fetal genes (G gamma and A gamma) intact. The size of these deletions has previously eluded exact definition. Using pulsed-field gel electrophoresis and the enzyme SfiI, which cuts only rarely in genomic DNA, we have constructed a large-scale restriction map of the beta-globin cluster in normal and HPFH DNA. The deletions in HPFH-1, which occurs in American blacks, and in HPFH-2, which occurs in Ghanaian blacks, are found to be approximately 105 kilobases (kb) in length, though the endpoints are staggered by approximately 5 kb. The fact that two previously reported gamma delta beta-thalassemia deletions to the 5' side of the beta-globin cluster are also about 100 kb suggests a common mechanism, possibly involving the loss of a complete chromatin loop.

Chromatin↗
PubMed1986

Stable integration of woodchuck hepatitis virus DNA in transplanted tumors and established tissue culture cells derived from a woodchuck primary hepatocellular carcinoma.

The fate of integrated woodchuck hepatitis viral (WHV) DNA was systematically investigated in DNA samples from primary hepatocellular carcinoma (HCC) of woodchucks, solid tumors transplanted in athymic mice derived from a primary HCC of woodchuck, and an established cell line of tissue culture originating from the transplanted tumor. In four of five woodchuck primary HCCs, WHV DNA integration was demonstrated in addition to various amounts of extrachromosomal replicative intermediate WHV DNA. The integration pattern of the primary HCCs does not indicate a common integration site on the host chromosome. The integration pattern in the established cells is identical to that in the transplanted tumor and similar but slightly different from that of the primary HCC. No extrachromosomal or replicative intermediates of WHV DNA were detected in the transplanted tumors or in the established cells of tissue culture. There are three integration sites on the chromosomes of the established cells. Results of Southern hybridization and restriction maps of cloned fragments suggest that all of these integrated WHV DNA sequences are not a complete genome but a part of the genome. A small portion corresponding to the cohesive region of the genome was not detected in all of these integrated WHV DNA. A positive role of WHV DNA integration on the generation of HCC is strongly suggested by the high incidence of WHV DNA integration in woodchuck primary HCCs and the stable maintenance of a certain mode of WHV DNA integration in the hepatoma-derived cell populations during passages of transplantation or serial growth of tissue culture.

Animals↗
PubMed1984

[Wide distribution of transposable phages in natural Pseudomonas aeruginosa populations].

Five phages (PH2, PH51, PH59, PH93 and PH132) which have some characteristics common with D3112, the transposable phage of Pseudomonas aeruginosa, were isolated from clinical P. aeruginosa isolates. The phages were distributed into 4 different immunity groups. The basic criteria used for selection of transposable phages have been: 1) Morphology of a phage particle, host range, similar inactivation with antiserum; 2) Similar sizes of phage genomes; 3) The presence of a variable non-phage nucleotide sequences covalently linked to phage genome DNA, which could be identified using restriction endonucleases or by heteroduplex analyses. The DNAs of the new phages are resistant to treatment with BamH1 endonuclease, like the DNAs of phages D3112, B39 and B3 described earlier. The restriction maps of the phage genomes are constructed.

Bacteriophages↗
PubMed1983

Direct transfer of the bacterial asparagine synthetase gene to mammalian cells.

Using specific mutants as a means of identification, the bacterial protein for asparagine synthetase (Asn Syn) was shown to be antigenically and electrophoretically similar to its mammalian counterpart. This observation prompted us to attempt direct transfer of the cloned bacterial gene for the enzyme to mammalian cells. DNA from the replicative form of clone M13 OriC, containing the bacterial gene for Asn Syn, was shown to be capable of causing transformation of Jensen rat Asn Syn- cells to cells capable of growth in Asn-free medium; no prior modification of the bacterial gene was required. This relatively inefficient transformation (20 colonies/micrograms DNA/10(6) cells) was sensitive or insensitive to restriction enzyme digestion of the M13 OriC DNA in complete agreement with the known restriction map of the bacterial gene. Clones of transformed rat cells contained the bacterial DNA, which was amplified if increased levels of the enzyme were demanded and lost if selection was removed. The clones also contained polysomal bacterial RNA and a new protein with properties similar but not identical to those of the bacterial enzyme. The biological significance of this unusual degree of compatibility between the prokaryotic and eukaryotic Asn Syn gene systems is discussed.

Animals↗
PubMed1992

Physical and genetic mapping of the genomes of five Mycoplasma hominis strains by pulsed-field gel electrophoresis.

We present the complete maps of five Mycoplasma hominis genomes, including a detailed restriction map and the locations of a number of genetic loci. The restriction fragments were resolved by field inversion gel electrophoresis or by the contour-clamped homogeneous-electric-field system of pulsed-field gel electrophoresis. All the ApaI, SmaI, BamHI, XhoI, and SalI restriction sites (total of 21 to 33 sites in each strain) were placed on the physical map, yielding an average resolution of 26 kb. The maps were constructed using three different approaches: (i) size determination of DNA fragments partially or completely cleaved with one or two restriction enzymes, (ii) hybridization analysis with purified restriction fragments and specific probes, and (iii) use of linking clones. A genetic map was constructed by hybridization with gene-specific probes for rpoA, rpoC, rrn, tuf, gyrB, hup, ftsY, the unc operon, the genes for two M. hominis-specific antigenic membrane proteins, and one gene encoding a protein with some homology to Escherichia coli alanyl-tRNA synthetase. The positions of mapped loci were partially conserved in the five strains except in one strain in which a 300-kb fragment was inverted. The numbers and order of mapped restriction sites were only partly conserved, and this conservation was restricted to certain regions. The gene order was compared with the gene order established for other bacteria and was found to be identical to that of the phylogenetically related Clostridium perfringens. The genome size of the M. hominis strains varied from 704 to 825 kb.

Blotting, Southern↗
PubMed1998

Physical mapping of the linear plasmid pSLA2-L and localization of the eryAI and actI homologs.

The 200-kb linear plasmid pSLA2-L was suggested to be involved in the production of lankamycin and lankacidin in Streptomyces rochei 7434AN4. In this study, we have constructed a physical map for 23 PstI fragments of pSLA2-L, the sum of which was 206 kb. Detailed restriction maps for both ends of pSLA2-L revealed the presence of terminal inverted repeats, the size of which was found to be 2.1 kb by cloning and sequencing of the end-points. Hybridization experiments using two polyketide biosynthetic genes, eryAI and actI, located their homologous regions on PstI fragments A and I, respectively.

Anti-Bacterial Agents↗
PubMed1995

Physical analysis of the tuberous sclerosis region in 9q34.

We report the construction of a physical map based on cloned DNA within the candidate region for the tuberous sclerosis complex (TSC1) gene on chromosome 9q34, between the markers D9S149 and D9S66. The DNA clones form three contigs consisting of 7 YACs, bridged by P1 and cosmid clones, and cover more than 950 kb of 9q34. Despite intensive screening of all available libraries, two gaps remain. A detailed physical map of much of this region was derived, and restriction mapping of the YAC, P1, and cosmid clones reveals novel CpG islands in this region. This set of genomic clones provides a resource for characterizing candidates for the TSC1 gene, guided by the location of CpG islands.

Base Sequence↗
PubMed1996

Sequence and transcription of Qa-2-encoding genes in mouse lymphocytes and blastocysts.

The protein product of the mouse preimplantation embryo development (Ped) gene, which controls the rate of preimplantation embryonic cleavage division and subsequent embryo survival, is the Qa-2 antigen. This major histocompatibility complex (MHC) class I b protein is encoded by four genes, Q6, Q7, Q8, and Q9. The present study was undertaken to begin to elucidate which of the four Qa-2-encoding genes are responsible for the Ped gene phenotype in the C57BL/6 mouse (H2(b)). First, restriction maps of the four genes, using 25 restriction enzymes, were created. The RE maps confirmed that Q6 is similar to Q8 and Q7 is similar to Q9, but that the Q6/Q8 gene pair differs from the Q7/Q9 gene pair. The genomic DNA sequences of Q6 and Q8 were determined, as well as the DNA sequences of exons 4 - 8 of Q9, and the 5' regulatory regions of Q6, Q8, and Q9. This DNA sequence information, combined with the published DNA sequence information for the entire Q7 gene and exons 1 - 3 of Q9, allowed us to design primers for reverse transcription-polymerase chain reaction that could distinguish which of the four genes were transcribed in mouse lymphocytes and embryos. It was found that all four genes are transcribed in lymphocytes, but only Q7 and Q9 are transcribed in mouse embryos. Thus, both Q7 and Q9 are candidates for the genes responsible for the Ped gene phenotype.

Amino Acid Sequence↗
PubMed1990

Characterization and comparison of mitochondrial DNAs and rRNAs from Penicillium urticae and P. chrysogenum.

Mitochondrial DNA (mt DNA) from a patulin producer, Penicillium urticae (synonym P. griseofulvum), was 27.8 kb +/- 0.6 kb in size by electron microscopy and 27.2 kb by agarose gel electrophoresis. Restriction endonuclease maps for nine restriction enzymes were constructed, and eleven fragments which covered the total range of the mt DNA were cloned into the Escherichia coli plasmid vector pUC19. Southern analysis of the native genomes of P. urticae and P. chrysogenum with six of the cloned fragments as probes indicated similar genome arrangements as well as similar restriction maps. Both the large and small rRNA genes of P. urticae and P. chrysogenum were located on these restriction maps using Southern hybridization, and the result also supported the similar arrangement. Agarose/formaldehyde gel electrophoresis indicated that the small rRNA was 1.5 kb in size in both species; but, surprisingly, the large rRNA was 4.2 kb in size for P. urticae and 3.5 kb for P. chrysogenum. These sizes were, respectively, 1.1 kb and 0.4 kb larger than those from the very closely related Aspergillus nidulans.

Blotting, Southern↗
PubMed1992

Characterization of a YAC and cosmid contig containing markers tightly linked to the myotonic dystrophy locus on chromosome 19.

Myotonic dystrophy (DM) is caused by a defect in an unknown gene that maps to 19q13.3, flanked by the tightly linked markers ERCC1 on the proximal side and D19S51 on the distal side. We report the isolation and characterization of overlapping YAC and cosmid clones around D19S51 for the construction of a physical map around this locus. The resulting contig contains the markers D19S51 and D19S62 (another new marker tightly linked to the DM locus) and the distal breakpoint of a radiation hybrid cell line used in the physical mapping of the DM region. We have compared the restriction maps of the YACs and cosmids with that of the genome to investigate the fidelity of these clones.

Base Sequence↗