Penicillinase plasmids of Staphylococcus aureus: restriction-deletion maps.
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Genes↗
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Rapid mapping of restriction sites of a DNA: restriction of agarose gel and two-dimensional analysis of end-labeled DNA.
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DNA Restriction Enzymes↗
Isolation, cloning and characterization of argF gene DNA from Escherichia coli K-12.
A 1650 base pair (BP) fragment carrying the entire argF structural gene with its associated control regions was isolated from an EcoRI/BamHI digest of phi80argFilambda cI857 DNA. This segment was cloned using the EcoRI and BamHI cleavage sites in the plasmid pBR322. A preliminary restriction map of the argF region was prepared. RNA polymerase binding studies indicated that the argF promoter is located approx. 30 base pairs from the EcoRI terminus of the cloned DNA segment.
Arginine↗
Physical localisation and cloning of the structural gene for E. coli initiation factor IF3 from a group of genes concerned with translation.
The structural genes for translational initiation factor IF3, threonyl-tRNA synthetase (TRS), the two subunits of phenylalanyl-tRNA synthetase (PRS), and a 12 000 mol. wt. protein of unidentified function are carried by the lambda p2 transducing phage. The localization of these genes on a restriction map of the Escherichia coli DNA insert was achieved by deletion mapping. In addition a set of plasmids carrying fragments of the original phage was constructed and helped to confirm these assignments. One plasmid, containing a 3.3 kb PstI fragment inserted into pBR322, does not code for any of the synthetase genes but causes strains carrying it to overproduce IF3.
Bacteriophage lambda↗
Molecular analysis of the human interferon-alpha gene family.
Fifteen DNA clones containing sequences related to human interferon-alpha cDNA were isolated from a human chromosomal gene bank (Nagata et al., Nature 287 (1980) 401-408) and characterized by restriction mapping, R-loop and heteroduplex analysis. Nine distinct DNA segments hybridized strongly with interferon-alpha 1 cDNA and formed R-loops with poly(A) RNA from interferon-producing human leukocytes; most if not all of these segments represent functional interferon genes. Five segments hybridized weakly with the probe and did not form R-loops with the poly(A) RNA; one of these was characterized as an interferon-alpha pseudogene. Several DNA segments overlap and define a region of 36 kilobase pairs (kb) that contains three strongly and three weakly hybridizing sequences. From our data and those of Goeddel et al. (Nature 290 (1981) 20-25) we conclude that there exist at least 11 distinct genes of gene-like sequences of the interferon-alpha type in the human genome, of which most likely represents an allelic variant, and at least five pseudogenes distantly related to the interferon-alpha genes.
Chromosome Mapping↗
Organization of the ribosomal RNA gene cluster in Aspergillus nidulans.
DNA coding for ribosomal RNA in Aspergillus nidulans was found to consist of a unit 7.8 kb in size which is tandemly repeated in the genome and codes for 5.8S, 18S and 26S rRNA. The repeat unit has been cloned, and its restriction map and the location of the individual rRNA coding sequences within the unit have been established.
Aspergillus nidulans↗
The human erythropoietin-encoding gene contains a CAAT box, TATA boxes and other transcriptional regulatory elements in its 5' flanking region.
We have reported the cloning and expression of a human erythropoietin (hEp)-encoding cDNA [Lee-Huang, Proc. Natl. Acad. Sci. USA 81 (1984) 2708-2712]. Using this hEp cDNA as a probe, we isolated a 9.3-kb BamHI genomic Ep clone from a human leukocyte library soon thereafter. The size and restriction map of this clone is in agreement with restriction analysis of human genomic DNA probed with the hEp cDNA, demonstrating that this clone is representative of the single hEp gene. This clone is unique in that it extends beyond any reported hEp genomic clone by 3.9 kb on the 5' side and by 1.8 kb on the 3' side. The promoter function of the newly described 5' flanking region has been demonstrated by the expression of biologically active hEp in transfected cells. We find that, despite reports to the contrary, hEp does contain classic canonical TATA boxes and a CAAT box. The 5'-flanking region also contains cytokine-responsive consensus sequences, tissue-specific and metal-responsive elements, CRE and GRE sites, and binding sites for transcription factors, including AP1, NF-kappa beta and Sp1. These regulatory elements have not been found in the hEp genomic clones thus far reported. The identification of these elements and their precise localization in hEp should be useful in studying the regulation of hEp expression, as well as in gene therapy and physiologic modulation of this hormone.
Activating Transcription Factor 2↗
Analysis of NotI linking clones isolated from human chromosome 3 specific libraries.
We have partially sequenced more than 1000 NotI linking clones isolated from human chromosome 3-specific libraries. Of these clones, 152 were unique chromosome 3-specific clones. The clones were precisely mapped using a combination of fluorescence in situ hybridization (FISH) and hybridization to somatic cell or radiation hybrids. Two- and three-color FISH was used to order the clones that mapped to the same chromosomal region, and in some cases, chromosome jumping was used to resolve ambiguous mapping. When this NotI restriction map was compared with the yeast artificial chromosome (YAC) based chromosome 3 map, significant differences in several chromosome 3 regions were observed. A search of the EMBL nucleotide database with these sequences revealed homologies (90-100%) to more than 100 different genes or expressed sequence tags (ESTs). Many of these homologies were used to map new genes to chromosome 3. These results suggest that sequencing NotI linking clones, and sequencing CpG islands in general, may complement the EST project and aid in the discovery of all human genes by sequencing random cDNAs. This method may also yield information that cannot be obtained by the EST project alone; namely, the identification of the 5' ends of genes, including potential promoter/enhancer regions and other regulatory sequences
Animals↗
Sequence organization and genomic complexity of primate theta 1 globin gene, a novel alpha-globin-like gene.
The alpha-like and beta-like globin genes have provided a paradigm for the study of molecular evolution and regulation of multigene families in eukaryotes. The human alpha-globin gene cluster, which is on chromosome 16 (ref. 1), consists of six genes arranged in the order 5'-zeta(embryonic)-psi zeta-psi alpha 2-psi alpha 1-alpha 2(adult)-alpha 1(adult)-3'. DNA sequencing data have demonstrated that zeta (ref. 6) and alpha 2 (or alpha 1, refs 7-9) are the embryonic and adult genes, respectively, while psi zeta (ref. 6), psi alpha 2 (ref. 5) psi alpha 1 (ref. 10) are all inactive pseudogenes. Restriction mapping analysis has shown that the structure of this locus in several anthropoid primates is nearly identical to that of the human. Recently, we have isolated the adult alpha-globin gene region from orang-utan, olive baboon and rhesus macaque by molecular cloning. We report here the complete nucleotide sequence of a gene located immediately downstream from the adult alpha 1-globin gene of the orang-utan, along with its flanking DNA. We designate this gene as theta 1, and show that it contains the essential sequence elements required for an expressive gene. The putative polypeptide is 141 amino acids long, identical to that of the alpha- or zeta-globin, but its predicted amino-acid sequence is nearly as different from the orang-utan alpha-globin (55 differences) as the human zeta-globin is from the human alpha-globin (59 differences), suggesting an ancient history for the theta 1-globin gene. Results of blot hybridization experiments using the cloned orang-utan theta 1 gene sequence as probe demonstrate a similar alpha 2-alpha 1-theta 1 linkage map existing in the human genome. Furthermore, multiple copies of sequences homologous to the theta 1 gene are detected in both human and orang-utan. These results cast a new light on the primate alpha-globin gene family, and have intriguing implications for the existence of previously unreported, functional globin-like gene(s) in the primate genomes.
Amino Acid Sequence↗
CD45; from alloantigen to mapping of restricted epitopes using recombinant soluble CD45 isoforms.
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Amino Acid Sequence↗
Moloney murine sarcoma virions synthesize full-genome-length double-stranded DNA in vitro.
Moloney murine sarcoma virus (MSV) virions incubated under optimal conditions were shown to support extensive synthesis of double-stranded DNA. The major product, a 5950-base-pair (6-kilobase-pair DNA) double-stranded DNA, was characterized by cleavage with restriction endonucleases and shown to contain a 600-nucleotide-long direct repeat at both ends of the MSV genome. Linear DNA molecules made in vivo shortly after infection were compared to the linear double-stranded DNA synthesized in vitro. The restriction maps of both viral DNA products were indistinguishable. The 600-base-pair repeat results in a progeny DNA molecule that is longer than the parental MSV genomic RNA. The generation of this repeat must involve a mechanism that allows the viral reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) to copy 5'- and 3'-terminal genomic (+) strand sequences twice.
Chromosome Mapping↗
Organization of the recA gene of Escherichia coli.
The restriction map of a BamHI DNA fragment that contains the recA gene of Escherichia coli has been established and a large portion of the fragment's nucleotide sequence has been determined. The coding region of the recA gene contains 1059 nucleotide residues and encodes a single protein of 353 amino acid residues. The amino acid sequence of the first five residues of the NH2 terminus of the recA protein agrees with the sequence predicted from the DNA sequence except for the absence of formylmethionine in the purified protein. Immediately after the coding sequence, there is a G+C-rich sequence with dyad symmetry followed by an A+T-rich sequence. These could signal termination of transcription. The site of initiation for synthesis in vitro of the recA messenger RNA has been determined by analysis of the 5' nucleotide sequence of [gamma-32P]ATP-labeled transcripts. The promoter region shos a high degree of symmetry and contains sequences commonly found in recognition and binding sites for RNA polymerase.
Amino Acid Sequence↗
Bacterio-opsin mutants of Halobacterium halobium.
The bacterio-opsin (bop) gene of Halobacterium halobium R1 has been cloned with about 40 kilobases of flanking genomic sequence. The 40-kilobase segment is derived from the (G+C)-rich fraction of the chromosome and is not homologous to the major (pHH1) or minor endogenous covalently closed circular DNA species of H. halobium. A 5.1-kilobase Pst I fragment containing the bop gene was subcloned in pBR322 and a partial restriction map was determined. Defined restriction fragments of this clone were used as probes to analyze the defects associated with the bop gene in 12 bacterio-opsin mutants. Eleven out of 12 of the mutants examined had inserts ranging from 350 to 3,000 base pairs either in the bop gene or up to 1,400 base pairs upstream. The positions of the inserts were localized to four regions in the 5.1-kilobase genomic fragment: within the gene (one mutant), in a region that overlaps the 5' end of the gene (seven mutants), and in two different upstream regions (three mutants). Two revertants of the mutant with the most distal insert had an additional insert in the same region. The polar effects of these inserts are discussed in terms of inactivation of a regulatory gene or disruption of part of a coordinately expressed operon. Given the defined nature of the bop mRNA-i.e., it has a 5' leader sequence of three ribonucleotides-these observations indicate that the bop mRNA might be processed from a large mRNA transcript.
Journal Article↗
Flow of mitochondrial DNA across a species boundary.
Restriction analysis shows that wild Scandinavian mice belonging to the species Mus musculus contain the mitochondrial DNA of a neighboring species, M. domesticus. This demonstration results from comparisons of Scandinavian mice with authentic M. domesticus and M. musculus from other parts of Europe. Electrophoretic and immunological analysis of eight diagnostic proteins confirms that mice from north of the hybrid zone in Denmark are M. musculus in regard to their nuclear genes. In contrast, the mice tested from this region and a nearby part of Sweden have exclusively M. domesticus types of mitochondrial DNA. Phylogenetic analysis of the restriction maps suggests that the mitochondrial DNAs found in Scandinavian M. musculus could stem from a single M. domesticus female.
Alleles↗
An improved FORTRAN 77 recombinant DNA database management system with graphic extensions in GKS.
We have improved an existing clone database management system written in FORTRAN 77 and adapted it to our software environment. Improvements are that the database can be interrogated for any type of information, not just keywords. Also, recombinant DNA constructions can be represented in a simplified 'shorthand', whereafter a program assembles the full nucleotide sequence from the contributing fragments, which may be obtained from nucleotide sequence databases. Another improvement is the replacement of the database manager by programs, running in batch to maintain the databank and verify its consistency automatically. Finally, graphic extensions are written in Graphical Kernel System, to draw linear and circular restriction maps of recombinants. Besides restriction sites, recombinant features can be presented from the feature lines of recombinant database entries, or from the feature tables of nucleotide databases. The clone database management system is fully integrated into the sequence analysis software package from the Pasteur Institute, Paris, and is made accessible through the same menu. As a result, recombinant DNA sequences can directly be analysed by the sequence analysis programs.
Algorithms↗
Structure and expression of myelin basic protein gene sequences in the mld mutant mouse: reiteration and rearrangement of the MBP gene.
The mld mutation on chromosome 18 in the mouse is a putative allele of the shiverer (shi) mutation. We have analyzed the structure of myelin basic protein (MBP) gene sequences in mld DNA by restriction mapping of genomic DNA. The results indicate that the mld chromosome carries two copies of the MBP structural gene, one of which is intact and one of which is interrupted. Genetic analysis indicates that the interrupted gene is close to the intact MBP structural gene and cosegregates with the mld mutation. We have also analyzed the levels of MBP polypeptides and MBP-specific mRNA in wild-type, homozygous and heterozygous shiverer and mld mice and in mice carrying both mutations. The results indicate that both shi and mld are cis-acting codominant mutations that cause severely reduced steady state levels of MBP-specific mRNA and MBP polypeptides in the brain. We have analyzed the total number of oligodendrocytes and the number of MBP-positive oligodendrocytes in mld and shi brain primary cultures. In shi cultures, none of the oligodendrocytes expresses MBP. However, in mld cultures, approximately 5% of the oligodendrocytes express MBP. The nature of the "revertant" mld oligodendrocytes is not known.
Animals↗
Structural variations in the Drosophila retrotransposon, 17.6.
More than 21 members of 17.6, a Drosophila retrotransposon, were isolated and their possible structural changes were examined by restriction mapping, blot hybridization, heteroduplex analysis and nucleotide sequence determination of long terminal repeats (LTRs). At least 7 members were found to suffer with terminal or internal long deletions. No pair of LTRs having an identical nucleotide sequence was found either within an element or between elements. Although an initiation site for the presumable genome-sized transcript of 17.6, a potential substrate for reverse transcription on translocation, was identified within the left-hand LTR, our results as a whole support the notion that the majority of 17.6s have continued to reside for a long period of time at their present chromosomal loci and hence the rate of translocation of 17.6 is very low.
Animals↗
Evidence for a unique human fibroblast interferon (IFN-beta 1) chromosomal gene, devoid of intervening sequences.
Direct restriction analysis of the human genome, using the Southern transfer technique and hybridization with a human fibroblast interferon (IFN-beta) complementary DNA insert probe, revealed the presence of a single gene; no additional closely related IFN-beta genes could be detected. A lambda-linked human gene library (Lawn et al., Cell, 15, 1157-1174 (1978)) was screened using the cDNA probe. Out of 600,000 recombinant phage examined, one single clone bearing interferon sequences was obtained. Restriction analysis of the relevant region revealed an identical restriction map as obtained for the IFN-beta 1 cDNA clones. No intervening sequences could be detected, either in the coding or the non-coding regions of the gene.
Base Sequence↗