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PubMed1989

Outbreak of poliomyelitis in South Africa investigated by oligonucleotide mapping.

An outbreak of paralytic poliomyelitis, due to polio type 1 virus, occurred in the Gazankulu region of the Transvaal in the Republic of South Africa in 1982. Thirty-four different isolates from this epidemic were characterized by oligonucleotide mapping. None of the isolates were shown to be vaccine related. Results suggest the existence of a number of apparently different wild-type viruses present in the area. The introduction of a single wild-type strain could not be clearly demonstrated. These findings have important implications for the management and control of future outbreaks.

Disease Outbreaks↗
PubMed1987

Structure and expression of a human gene coding for a 71 kd heat shock 'cognate' protein.

In all eukaryotes examined so far, hsp70 gene families include cognate genes (hsc70) encoding proteins of about 70 Kd which are expressed constitutively during normal growth and development. We have investigated the structural relationship of heat-inducible and cognate members of the human hsp70 gene family. Among several human genomic clones isolated using Drosophila hsp/hsc70 probes, one contained an hsc70 gene. Its complete sequence is reported here. It is split by eight introns and encodes a predicted protein of 70899 d that would be 81% homologous to hsp70. Structural comparisons with corresponding genes from other species provide one of the most striking examples of gene conservation. Isolation of a corresponding cDNA clone, RNA-mapping and in vitro translation data demonstrate that the gene is expressed constitutively and directs the synthesis of a 71 kd protein. The latter is very likely to be identical to a clathrin uncoating ATPase recently identified as a member of the hsp70-like protein family.

Amino Acid Sequence↗
PubMed1990

In vitro transcription and DNA binding characteristics of chloroplast and etioplast extracts from mustard (Sinapis alba) indicate differential usage of the psbA promoter.

The psbA gene which is differentially expressed in vivo in chloroplasts and etioplasts has an unusual promoter, containing both prokaryotic-type '-35' and '-10' elements and a sequence motif that resembles the nuclear TATA box. Single base pair substitutions were introduced into the mustard psbA promoter and the mutants were tested in transcription and DNA binding experiments, using extracts from either chloroplasts or etioplasts. Positions within the '-35' region appear to play an essential role in the chloroplast but not in the etioplast system. Altering the first or second position of the 'TATA box'-like region led to decreased psbA in vitro transcription in either plastid extract. These two mutations, however, did not affect binding of extracts to the (linear) psbA promoter fragment in gel retardation assays. Fragments carrying two other plastid promoters effectively competed psbA promoter binding of the etioplast extract, but more weakly that of the chloroplast extract. Lambda exonuclease mapping shows that the 5' border of the binding region is more upstream with the etioplast than with the chloroplast system, whereas the 3' border appears to be the same. Hence, protein(s) of the two plastid types seem to interact differently with the mustard psbA promoter in vitro and perhaps also in vivo.

Base Sequence↗
PubMed1994

The exon structure of the mouse alpha 2(IX) collagen gene shows unexpected divergence from the chick gene.

One cosmid and two overlapping phage clones covering the entire mouse alpha 2(IX) collagen gene including 12 kilobase pairs (kb) of 5'- and 8 kb of 3'-flanking sequences were isolated from two genomic libraries. The overall gene structure was determined by restriction mapping and nucleotide sequencing. The gene spans 16 kb from the start of transcription to the polyadenylation site and contains 32 exons. It codes for a mRNA of 3 kb that translates into a polypeptide of 688 amino acids. The intron-exon junctions and mRNA structure were confirmed by amplification of cDNA made for mouse cartilage RNA. The coding sequence of the mouse alpha 2(IX) collagen gene shows marked similarities to those for other type IX collagen chains. Although the overall exon-intron organization of the mouse gene is very similar to the chick alpha 2(IX) gene, some unexpected differences were observed at the splice junctions. Split codons characteristic for the central triple helical domain of the chick were not found in the mouse gene that thus exhibited a long stretch of exons with sizes that are multiples of 9 base pairs in this domain. The promoter of the mouse alpha 2(IX) collagen gene contains some G + C-rich elements including three Sp1 consensus recognition sites and a far upstream CCAAT box but no TATAA box. Both primer extension and RNase protection assays revealed several transcription start sites within 418 base pairs of the promoter. The present study reports the first complete nucleotide sequence of any type IX collagen gene and forms the basis for comparative structural studies on this collagen type and for experiments involving transgenic mice.

Amino Acid Sequence↗
PubMed1980

Localization of the replication control region on the physical map of the octopine Ti plasmid.

A series of small plasmids has been derived from the octopine plasmid pTi-B6 by in vitro manipulation. The smallest plasmid that is able to replicate in Agrobacterium tumefaciens contains the ampicillin-resistance determinant from Tn1, coupled to a piece of DNA that is homologous to HpaI fragment number 11 of the octopine Ti plasmid pTi-Ach 5. The incompatibility functions are also specified by this region.

Arginine↗
PubMed1985

Molecular cloning and physical mapping of the Neurospora crassa 74-OR23-1A mitochondrial genome.

To provide for thorough sampling of the Neurospora crassa mitochondrial genome for evolutionary studies, recombinant plasmids containing each of the EcoRI digestion fragments of the genome were assembled and used to map the locations of 89 additional restriction endonuclease cleavage sites, representing 10 newly mapped enzymes and 2 previously unmapped HincII sites. Data used to locate new restriction sites were obtained from digestions of whole mitochondrial DNA, digestions of the cloned EcoRI mitochondrial DNA fragments and hybridizations between new restriction fragments and the cloned fragments. Length measurements of the total genome and of EcoRI fragment 1 are larger than commonly reported.

Cloning, Molecular↗
PubMed1990

Genetic drift of equine 2 influenza A virus (H3N8), 1963-1988: analysis by oligonucleotide mapping.

Comparative analysis by RNA oligonucleotide fingerprints of total genomic RNA as well as the individual RNA segments of equine 2 influenza A virus strains from 1963, 1968, 1979, 1984, 1987 and 1988 revealed genetic diversity. Strains from the epizootic outbreak during 1978-1979 showed minor differences among their genomes. The Swedish isolates from 1979 up to 1988 showed increasing genomic heterogeneity indicating genetic drift.

Animals↗
PubMed1988

AsnC, a multifunctional regulator of genes located around the replication origin of Escherichia coli, oriC.

The expression of the gidA gene which is located immediately counterclockwise of the replication origin of Escherichia coli, oriC, was found to be negatively regulated by the AsnC protein in an in vitro transcription-translation system. This effect is not due to simple repression of transcription originating at the gidA promoter, because the AsnC protein did not change the level of gidA promoter dependent transcription as analysed by promoter-galK fusions and by S1 mapping. From these data we conclude that the AsnC protein controls gidA gene expression at a post-transcriptional level. gidA is the third gene in the oriC region, besides asnA and asnC, whose expression is under AsnC control. However, the mechanisms involved are different: regulation of transcription in the case of asnA and asnC and post-transcriptional control of gidA. The gidA promoter was mapped by deletion analysis and by S1 mapping. We defined two regions that affect promoter activity negatively. Additional transcripts, regulated by AsnC, started more than 300 bp upstream of the gidA promoter and were found to enter the gidA region. These transcripts, originating either at the mioC and/or the ansC promoter traverse the replication origin.

Asparaginase↗
PubMed1989

Mapping of monoclonal antibodies to human factor IX.

We used recombinant DNA techniques to map a panel of six monoclonal antibodies (MoAbs) to regions of the human factor IX molecule. A-2 maps to 17 amino acids at the amino terminus of the heavy chain of IXa; 2D5, an inhibitor of clotting, is defined to 36 amino acids of the first EGF-like domain of human factor IX. A-4, A-5, C10D, and FXC008 all map to a region of the heavy chain containing amino acids 180 through 310, suggesting an immunodominant site. FXC008 has been reported to interfere with binding of factor IXa to factor VIII:Ca.

Amino Acid Sequence↗
PubMed1988

Transcriptional analysis of Bacillus subtilis rRNA-tRNA operons. II. Unique properties of an operon containing a minor 5 S rRNA gene.

This is part of a series of two papers on gene regulation in Bacillus subtilis rRNA-tRNA operons that contain large clusters of tRNA genes. The preceding paper (Vold, B.S., Okamoto, K., Murphy, B.J., and Green, C.J. (1988) J. Biol. Chem. 263, 14480-14484) investigates the rrnB operon containing 21 tRNA genes, and this paper investigates a B. subtilis rRNA-tRNA operon containing 16 tRNA genes and a minor 5 S rRNA. Hybridization studies suggest this minor 5 S rRNA occurs as a single copy in the B. subtilis 168 genome. S1 nuclease mapping indicates that this minor 5 S rRNA gene has its own promoter. No promoters have been found immediately 5' to any of the major 5 S rRNA species in B. subtilis rRNA operons. S1 mapping of the spacer region between the 23 S and minor 5 S rRNA revealed that the maturation of the 23 S rRNA in this operon may arise from an unusual processing mechanism. S1 nuclease mapping experiments suggest the existence of a promoter element immediately upstream of the last gene, for tRNA(Leu CAA), in the operon. A precursor leucine tRNA resulting from transcription of this last tRNA gene was observed in Northern hybridizations, and the amounts of this precursor increased during sporulation. A single terminator-like element is located just upstream of this last tRNA gene; however, S1 nuclease mapping experiments suggest that some read-through transcription occurs. Thus, all 16 tRNA genes are under control of the upstream 16 S rRNA promoters and the minor 5 S rRNA promoter. However, the last tRNA gene is primarily under the control of its own unique promoter.

Bacillus subtilis↗
PubMed1997

Determination of specific DNA strand discontinuities with nucleotide resolution in exponentionally growing bacteria harboring rolling circle-replicating plasmids.

Plasmid replication by the rolling circle mechanism and conjugative transfer of plasmids require the generation of a specific strand discontinuity in the DNA. In both processes cleavage at the so-called nic site is catalyzed by plasmid-encoded proteins. The strand discontinuities at the conjugative origins of transfer of plasmid pE194 and pMV158 were determined in Bacillus subtilis and Streptococcus pneumoniae, respectively, with a recently developed runoff DNA synthesis assay. The positions of intracellular cleavage within the respective transfer origins were shown to coincide with the site predicted for pE194 and with the nic site determined in vitro for pMV158. For pMV158, the influence of a mutation in the S. pneumoniae polA gene on the efficiency of replication was investigated. In addition, the nic site within the double-stranded origin of the-rolling circle-replicating plasmid pMV158 in S. pneumoniae as well as that of pFX2 in Escherichia coli was mapped with nucleotide resolution.

Bacillus subtilis↗
PubMed1986

Molecular cloning and sequencing of a gene encoding biologically active porcine alpha-interferon.

Nine distinct genomic clones containing human alpha 1-interferon (IFN-alpha 1) related sequences were isolated from a porcine genomic library constructed in phage lambda. Restriction mapping and Southern blot analysis revealed that these clones contained a total of 10 potential porcine IFN-alpha genes or pseudogenes belonging to a multigene family of at least 12 members. One of these genes was subcloned in plasmid pUC8 and the recombinant plasmid obtained was shown to direct the synthesis of a low but detectable IFN-alpha activity in Escherichia coli JM103. The sequence of this porcine IFN-alpha gene (Po IFN-alpha 1) was determined. As expected, it contained no introns and encoded a 189-amino-acid-long preprotein with a putative signal peptide of 23 residues. The homology to human (Hu)IFN-alpha 1 was 78.5% at the nucleotide level and 64% at the amino acid level.

Amino Acid Sequence↗
PubMed1987

High-resolution mapping of DNase I hypersensitive site in the chicken beta-tubulin gene transfected into monkey cells.

In this report, we describe a method for high-resolution mapping of a nuclease hypersensitive site (HS) at the promoter region of the chicken beta 2-tubulin gene transfected into monkey Cos-7 cells. Using the indirect end-labeling method of Wu, a tissue-specific nuclease HS was located initially approximately 100 base pairs upstream of the transcription start site. For high-resolution mapping of the HS in the transfected DNA, a DNA fragment of about 400 base pairs containing the promoter region of the beta 2 gene was subcloned into M13 phage. A single-stranded DNA probe with a defined restriction enzyme cleavage site at one end was prepared from the M13 phage DNA template. The single-stranded probe and the restriction enzyme-digested DNA, prepared from nuclease-digested nuclei from transfected cells, were sequentially annealed back to the template. After ligation between the probe and the transfected DNA, the HS was determined by DNA sequencing gel electrophoresis. This method permits determination of the 3' boundary of the HS with precision at the single-nucleotide level. We mapped a major HS at -107 nucleotides of the beta 2-tubulin gene, where a CACCC-like sequence is present. The application of this method in determining HS is discussed.

Animals↗
PubMed1991

Cloning, expression, and nucleotide sequence of rat liver sterol carrier protein 2 cDNAs.

The structure and expression of rat sterol carrier protein 2 (SCP2) were studied by cloning and sequencing SCP2 cDNAs and by hybridizing the SCP2 cDNA to RNA from a variety of rat tissues. Initial screening of a rat liver cDNA library with an oligonucleotide probe derived from the rat SCP2 protein sequence revealed an 825-base pair cDNA clone coding for the complete SCP2 protein sequence. Our results suggest that rat liver SCP2 is 143 amino acid residues long, including a 20-amino acid residue prepeptide (pre-SCP2). Using the rat SCP2 cDNA as a probe for Northern blot hybridization analyses under high stringency conditions with rat liver poly(A) RNA, four mRNA species differing in their sizes and levels of expression are detected. The lengths of the corresponding mRNAs are 0.8, 1.4, 2.1, and 2.7 kilobases (kb), respectively. In all other tissues analyzed mRNAs hybridizing to the SCP2 cDNA probe were detected in levels much lower than in the liver. In these tissues the vast majority of the expressed SCP2 mRNAs consists of the 0.8-kb mRNA. Results from Southern blotting hybridization analyses with rat genomic DNA suggest that the four different mRNA species are products of a single gene. Extensive screening of rat liver cDNA libraries using rat liver SCP2 cDNA probes led to the isolation of 10 additional clones, all of them containing cDNA insertions longer than 0.8 kb. Restriction mapping and nucleotide sequencing analyses of these cDNAs indicate that eight extend upstream of the SCP2 cDNA, and two extend 5' as well as 3'. Translation of the sequence extending most upstream suggests that this cDNA is a nearly full-length cDNA coding for a protein of 547 amino acids (SCP chi). SCP chi contains a 404-amino acid amino-terminal extension as compared with pre-SCP2 whereas its carboxyl-terminal part is identical with pre-SCP2. Sequencing of the 3' extension cDNAs suggests that polyadenylation had occurred 651 nucleotides downstream as compared with the remaining cDNAs. A probe from this region hybridizes to the 1.4- and 2.7-kb mRNAs. From our data we conclude that the 1.4- and 2.7-kb mRNA species are generated by alternative polyadenylation of the 0.8- and 2.1-kb mRNAs, respectively. In contrast, the 2.1- and 2.7-kb mRNAs contain an approximately 1300-nucleotide extension upstream of the 0.8- and 1.4-kb mRNAs, suggesting differential splicing of the primary SCP2 gene transcript leading to expression of pre-SCP2 and SCP chi from a single gene.

Amino Acid Sequence↗
PubMed1987

Multiple c-myb transcript cap sites are variously utilized in cells of mouse haemopoietic origin.

Mouse c-myb gene transcripts in various cells of haemopoietic origin were analysed using S1 nuclease and RNase mapping techniques and by Northern blotting. It was found that the prevalent 3.8-kb c-myb mRNA present in thymocytes, T cell leukaemias, myelomonocytic leukaemias, erythroleukaemias and myeloid stem cells was initiated at several cap sites mapping within a region 97-244 bp upstream from the protein coding sequence. Utilization of additional cap sites mapping further upstream was also observed in certain cells, most notably thymocytes, and this gave rise to RNA species (4.3-5.6 kb) larger than the presumptive mRNA. In contrast, myeloma cell c-myb transcripts, which are much less abundant than those in more immature haemopoietic cells, were found to be initiated at a restricted set of cap sites mapping 244-277 bp upstream of the coding sequence. Hence, these data suggest that the abundance of the c-myb mRNA may be regulated by a process involving selective utilization of mRNA cap sites. Sites hypersensitive to DNase I were associated with mRNA cap sites in cells that expressed c-myb.

Animals↗
PubMed1986

Restriction enzyme map of cryptic plasmid accompanying pR711b and pR409; identity of pR409 and pR388.

A reference strain containing the IncW plasmid, pR409, was found to contain also a cryptic, conjugative plasmid, here named pJR15. After the separation of the two plasmids into different strains of bacteria, pR409 was found to have an identical restriction enzyme map to the IncW plasmid, pR388. Both pR409 and pR388 were isolated from the same hospital in London and confer sulphonamide and trimethoprim resistance. Originally pR409 was reported to confer resistance to tetracycline, but this was not confirmed by the Plasmid Reference Center. pR409 appears to represent another isolate of pR388 in a different host background. The restriction enzyme map of the cryptic plasmid, pJR15 (39 kb), has been determined. pJR15 was found compatible with plasmids from 15 different incompatibility groups and has been found also in the reference strain of the IncD plasmid, pR711b. The HindIII and BglII digests of pR711b are shown. The possible presence of the conjugative plasmid, pJR15, should be examined for studies on the sex pili or chromosomal mobilization properties of pR711b.

DNA Restriction Enzymes↗
PubMed1999

A hydrogen-bonding triad stabilizes the chemical transition state of a group I ribozyme.

BACKGROUND: The group I intron is an RNA enzyme capable of efficiently catalyzing phosphoryl-transfer reactions. Functional groups that stabilize the chemical transition state of the cleavage reaction have been identified, but they are all located within either the 5'-exon (P1) helix or the guanosine cofactor, which are the substrates of the reaction. Functional groups within the ribozyme active site are also expected to assist in transition-state stabilization, and their role must be explored to understand the chemical basis of group I intron catalysis. RESULTS: Using nucleotide analog interference mapping and site-specific functional group substitution experiments, we demonstrate that the 2'-OH at A207, a highly conserved nucleotide in the ribozyme active site, specifically stabilizes the chemical transition state by approximately 2 kcal mol-1. The A207 2'-OH only makes its contribution when the U(-1) 2'-OH immediately adjacent to the scissile phosphate is present, suggesting that the 2'-OHs of A207 and U(-1) interact during the chemical step. CONCLUSIONS: These data support a model in which the 3'-oxyanion leaving group of the transesterification reaction is stabilized by a hydrogen-bonding triad consisting of the 2'-OH groups of U(-1) and A207 and the exocyclic amine of G22. Because all three nucleotides occur within highly conserved non-canonical base pairings, this stabilization mechanism is likely to occur throughout group I introns. Although this mechanism utilizes functional groups distinctive of RNA enzymes, it is analogous to the transition states of some protein enzymes that perform similar phosphoryl-transfer reactions.

Animals↗
PubMed1988

Transcriptional terminators in the caa-cal operon and cai gene.

We have analysed by S1 nuclease mapping the in vivo termination sites of transcription of the caa-cal operon and cai gene. The termination region for caa mRNA (T1A terminator) features characteristics of a rho-independent terminator. This terminator is a convergent transcription terminator, its complementary secondary structure being present at the 3'-end of cai mRNA. The caa-cal mRNA terminator (T2A terminator) has a stable potential secondary structure and shows homology with rho-dependent terminators. In vitro transcription of caa-cal operon demonstrated that the two terminators T1A and T2A are efficient. The 3'-ends of the mRNAs which end at T1A and T2A were analysed by S1 mapping with total RNA purified from a mutant strain deficient in exoribonuclease activities, in particular RNase II. The results suggest that the potential secondary structures of T1A and T2A are sufficiently stable to prevent 3'-end degradation by RNase II. On the other hand, the T2A terminator should be efficient enough to stop transcription through the downstream DNA region involved in pColA replication.

Base Sequence↗