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PubMed1990

Tandemly duplicated Caenorhabditis elegans collagen genes differ in their modes of splicing.

Caenorhabditis elegans contains 50 to 150 collagen genes dispersed throughout its genome. We have determined the complete nucleotide sequences of two collagen genes, col-12 and col-13, that are separated by only 1800 bases and are transcribed in the same direction. The 951 nucleotides of their coding regions differ by only five nucleotides (99.5% identity). The amino acid sequences are identical except for two conservative amino acid changes within the putative secretory signal sequences, so the mature forms of the col-12 and col-13 collagens would be identical. The position and sequence of the intron (52 base-pairs) within the coding region of each gene are perfectly conserved. In contrast to the coding regions and the introns, the 5' and 3' flanking regions show little sequence similarity, col-12 and col-13 are expressed at similar levels at the same developmental stages, and appear to utilize conserved TATA boxes and transcription start sites. The major differences between the genes is that, preceding the initiator ATG, col-12 has a cis-spliced intron, while col-13 is transspliced. Thus, col-12 and col-13 are essentially identical in all aspects except that the col-12 mRNA has a 26-nucleotide cis-spliced leader at the same place where the col-13 mRNA has a 22-nucleotide trans-spliced leader. These results suggest that col-12 and col-13 are derived from a gene duplication and that sequence homology in the coding regions, but not in the flanking regions, has been maintained by gene conversion. The fact that the only significant difference between the two genes is in their modes of splicing suggests that cis and trans-splicing can be interchanged during gene evolution.

Amino Acid Sequence↗
PubMed1991

In vitro assembly of a positioned nucleosome near the hypersensitive region in simian virus 40 chromatin.

Previous studies have identified a nucleosome near a potential late boundary for the nuclease-hypersensitive region in simian virus 40 chromatin. We have performed in vitro reconstitution analysis to determine whether the underlying DNA sequence encodes for the assembly of this nucleosome and applied hydroxyl radical and DNase I footprinting techniques to examine the structure of the reconstituted nucleosome. Both methods revealed the formation of a precisely positioned nucleosome in vitro, on a fragment spanning the strong in vivo nucleosome location site determined previously in the viral chromatin. The center of the positioned nucleosome maps between nucleotide 384 and 387 on simian virus 40 DNA. The corresponding nucleosome core includes the major-late transcription site (12 base-pairs within the core), the MspI site, and a segment shown previously to adopt a bent structure in the absence of proteins. The hydroxyl radical produces a strikingly well-defined cleavage pattern over the bent DNA incorporated in nucleosomes. The dominant periodicity of DNA in this nucleosome is 10.26 base-pairs per turn. The distribution of the .OH cut sites in the positioned nucleosome provides strong support for models in which the minor grooves of the A/T-rich tracts are oriented toward the histone core while the minor grooves of the G/C-rich sequences are facing outward.

Base Sequence↗
PubMed1991

Initiation of the UvrABC nuclease cleavage reaction. Efficiency of incision is not correlated with UvrA binding affinity.

The incision steps of Escherichia coli nucleotide excision repair are mediated by the UvrABC nuclease complex. We have previously shown that the UvrABC nuclease specifically incises apyrimidinic (AP) sites less efficiently than o-benzylhydroxylamine-modified apyrimidinic (BA) sites. To investigate these differences, quantitative DNase I footprinting titration studies were performed. The UvrA binding isotherms were similar for both the AP site (Kd = 6 x 10(-9) M) and the bulkier BA lesion (Kd = 14 x 10(-9) M), despite the fact that the extent of incision differs for these two lesions. It was also found that the relative binding affinity of the preincision UvrA2B complex to the AP and BA substrates differs significantly with estimated apparent equilibrium dissociation constants (Kd) of 4 x 10(-9) M and 80 x 10(-9) to 120 x 10(-9) M, respectively. These results indicate that incision efficiency does not correlate to UvrA binding affinity, but is a direct result of interactions between the UvrA2B complex and the site of the DNA damage. It is also shown that high UvrA concentrations are inhibitory to the UvrABC nuclease reaction.

Base Sequence↗
PubMed1991

Synthetic DNA bending sequences increase the rate of in vitro transcription initiation at the Escherichia coli lac promoter.

Appropriately phased DNA bending sequences replacing the CAP binding site upstream from the lac promoter increase by roughly tenfold the rate of specific transcription initiation from a superhelical promoter template in vitro; promoter occlusion results from polymerase binding to the upstream (dA)n.(dT)n tracts, but this phenomenon is not responsible for the observed phase-dependent transcriptional activity. The rates of open complex formation at both P1 and P2 promoters respond in a similar phase-dependent way to the synthetic curved DNA sequences.

Base Sequence↗
PubMed1991

Identification of the different intermediates in the interaction of (A)BC excinuclease with its substrates by DNase I footprinting on two uniquely modified oligonucleotides.

(A)BC excinuclease is the enzymatic activity resulting from the joint actions of UvrA, UvrB and UvrC proteins of Escherichia coli. The enzyme removes from DNA many types of adducts of dissimilar structures with different efficiencies. To understand the mechanism of substrate recognition and the basis of enzyme specificity, we investigated the interactions of the three subunits with two synthetic substrates, one containing a psoralen-thymine monoadduct and the other a thymine dimer. Using DNase I as a probe, we found that UvrA makes a 33 base-pair footprint around the psoralen-thymine adduct and that UvrA-UvrB make a 45 base-pair asymmetric footprint characterized by a hypersensitive site 11 nucleotides 5' to the adduct and protection mostly on the 3' side of the damage. Conditions that favor dissociation of UvrA from the UvrA-UvrB-DNA complex, such as addition of excess undamaged DNA to the reaction mixture, resulted in the formation of a 19 base-pair UvrB footprint. In contrast, a thymine dimer in a similar sequence context failed to elicit a UvrA, a UvrA-UvrB or UvrB footprint and gave rise to a relatively weak DNase I hypersensitive site typical of a UvrA-UvrB complex. Dissociation of UvrA from the UvrA-UvrB-DNA complex stimulated the rate of incision of both substrates upon addition of UvrC, leading us to conclude that UvrA is not a part of the incision complex and that it actually interferes with incision. The extent of incision of the two substrates upon addition of UvrC (70% for the psoralen adduct and 20% for the thymine dimer) was proportional to the extent of formation of the UvrA-UvrB-DNA (i.e. UvrB-DNA) complex, indicating that substrate discrimination occurs at the preincision step.

Base Composition↗
PubMed1992

Mutations that eliminate the requirement for the vertex protein in bacteriophage T4 capsid assembly.

The capsid of bacteriophage T4 is composed of two essential structural proteins, gp23, the major constituent of the capsid, and gp24, a less prevalent protein that is located in the pentameric vertices of the capsid. gp24 is required both to stabilize the capsid and to allow it to be further matured. This requirement can be eliminated by bypass-24 (byp24) mutations within g23. We have isolated, cloned and sequenced several new byp24 mutations. These mutations are cold-sensitive in the absence of gp24, and are located in regions of g23 not known to contain any other mutations affecting capsid assembly. The cold-sensitivity of the byp24 mutations can be reduced by further mutations within g23 (trb mutations). Cloning and sequencing of these trb mutations has revealed that they lie in regions of g23 that contain clusters of mutations that cause the production of high levels of petite and giant phage (ptg mutations). Despite the proximity of the trb mutations to the ptg mutations, none of the ptg mutations has a Trb phenotype. The mutation ptE920g, which is also located near one of the ptg clusters, and which produces only petite and wild-type phage, has been shown to confer a Trb but not a Byp24 phenotype. The relevance of these observations to our understanding of capsid assembly is discussed.

Alleles↗
PubMed1989

Abnormal glucocorticoid receptor gene and mRNA in primary cortisol resistance.

Abnormal steroid hormone receptors have been implicated as causing several forms of primary steroid hormone resistance in humans, but as yet no abnormality has been described at the gene level. We describe the analysis of the mRNA and genomic DNA from the Epstein-Barr (EB) virus transformed cells of two siblings with Primary Cortisol Resistance. The cells of the propositus and his brother show a decreased level of glucocorticoid receptor (GR) mRNA, and the genomic DNA of both individuals shows an altered restriction enzyme pattern with the restriction enzyme Bgl II, one of eleven restriction enzymes tested. The genomic differences could be detected with a probe specific for the putative steroid binding domain of the human GR gene.

Cell Transformation, Viral↗
PubMed1991

Deletions of mitochondrial DNA in Kearns-Sayre syndrome and ocular myopathies: genetic, biochemical and morphological studies.

Genetic, biochemical and morphological investigations were conducted on skeletal muscle mitochondria from 6 cases of ocular myopathy: 4 cases with Kearns-Sayre syndrome (KSS) and 2 with chronic progressive external ophthalmoplegia. All of these 6 cases showed mitochondrial DNA (mtDNA) deletions in addition to normal sized DNA in the quadriceps muscle. The deletions ranging from 3 to 8 kbp were also mapped between nucleotides 5500 and 16000 by Southern blot. The deleted genes encoded for some subunits of complexes I, IV, V and 5-10 tRNAS. The boundaries of the deletions have been sequenced in three patients. Five patients had mitochondrial respiratory chain deficiency in complex I as shown by the low oxygen consumption in isolated mitochondria using three NAD(+)-linked substrates. Mitochondria with an abnormal ultrastructure were also observed in 2 cases. A good relationship between the cytochrome c oxidase deficiency and the amount of deleted mtDNA was shown in our present investigations.

Adult↗
PubMed1988

Characterization of the types of mutational events that spontaneously occur in a plasmid system.

The immunity region from a cI857 derivative of bacteriophage lambda has been cloned into the EcoRI site of pBR322 to produce a plasmid that can be used to analyze spontaneous mutagenesis. Cells containing this plasmid are temperature-sensitive for growth unless mutations have occurred that somehow prevent the expression of the kil gene in the lambda fragment at non-permissive temperature. 678 such temperature-resistant mutants from 10 independent subcultures each of 2 different recA- E. coli strains have been collected, and the nature of the plasmid mutations obtained has been analyzed. All of the subcultures contained mutants that allowed growth at the restrictive temperature without showing a detectable change in plasmid size. 75% of the total mutants fell in this class. More than half of these mutations involved the lambda leftward promoter, pL, and such mutants were found in all 20 subcultures. The remaining 25% of the mutations involved a change in plasmid size and mutations of this class were found in 18 of 20 subcultures. 12% of the total mutants (found in 16 of 20 subcultures) had an insertion of IS1 in the region between pL and the lambda kil gene. 6% of the total mutants had undergone an IS1-mediated deletion, while 1% were mixed colonies in which multiple IS1-mediated events had occurred. About 1% of the total mutants had undergone complex IS1-mediated DNA rearrangement(s) that have not yet been characterized. In total, 11 of 20 subcultures yielded isolates where IS1-mediated rearrangements had occurred. The remaining 4% of the mutations included insertions of IS5, IS30, and an IS1 family member that appears to be IS1T as well as IS1T-mediated deletions and deletions that do not appear to have been mediated by any insertion sequence. A mutant with both an IS1 insertion and an alteration involving pL has also been isolated.

Bacteriophage lambda↗
PubMed1985

Defining the borders of the chicken proto-fps gene, a precursor of Fujinami sarcoma virus.

The transforming (onc) genes of retroviruses contain specific sequences, derived from as yet poorly defined, normal cellular genes, termed proto-onc genes. Proto-onc genes must be defined to explain their docility compared to the oncogenicity of the viral derivatives. Here we set out to determine the borders of the chicken proto-fps gene from which the onc genes of avian Fujinami (FSV) and PRC sarcoma viruses (PRCSV) are derived. These onc genes are hybrids of an element from the gag gene of retroviruses (delta gag) linked to a 2.8-kb domain from proto-fps. To identify the 5' border of proto-fps we have sequenced 1.5 kb beyond the 5' border of overlap with viral fps utilizing a proto-fps clone derived previously. A possible promoter was identified that maps 736 nucleotides from this border. The 736 nucleotides contain two possible exons with 121 codons, and short regions of homology with the delta gag termini of FSV and PRCII. A translation stop codon and an adjacent polyadenylation signal were identified just prior to the 3' border of overlap with viral fps within a 1.15-kb sequence of a newly isolated proto-fps clone. Comparing four exons within this 1.15 kb proto-fps sequence with known fps equivalents of FSV and PRCSV, we have detected strain-specific, but no common point mutations in each viral genome. A 3.3-kb polyadenylated proto-fps mRNA was detected in chicken liver RNA by gel electrophoresis and hybridization with proto-fps DNA. We conclude that the coding capacity of proto-fps is just over 3 kb, consistent with the size of the putative proto-fps protein of 98 kDa and hence slightly larger than that of viral fps. Thus proto-fps and the viral delta gag-fps genes each contain distinct 5' regulatory and coding sequences and share the 3' terminal fps domains. It is suggested that this difference, rather than scattered point mutations, is responsible for the oncogenic function of the viral genes and the unknown cellular function of proto-fps.

Animals↗
PubMed1987

Geographic distribution of wild poliovirus type 1 genotypes.

Determination of the patterns of genomic variation among RNA virus isolates is a powerful approach for establishing their epidemiologic interrelationships. The standard technique for such studies, ribonuclease T1 oligonucleotide fingerprinting, can detect similarities only among very closely related isolates. The rapid evolution of the poliovirus genome during transmission in humans requires the application of alternate methods to identify more distant relationships. To obtain a substantially broader view of the distribution of wild poliovirus type 1 genotypes in nature, we compared 150 bases of genomic sequence information (encoding parts of the capsid protein VP1 and the noncapsid protein 2A) from 62 isolates obtained from poliomyelitis patients in five continents. The partial sequence information allowed us to (1) identify numerous geographic foci of endemic circulation of wild type 1 polioviruses, (2) reveal previously unsuspected links between cases in distant communities, (3) monitor the displacement from the environment of preexisting polioviruses by viruses from other regions, and (4) recognize the recombinant (vaccine-wild; wild-wild) origins of some epidemic polioviruses.

Base Sequence↗
PubMed1987

Studies on the recombination between RNA genomes of poliovirus: the primary structure and nonrandom distribution of crossover regions in the genomes of intertypic poliovirus recombinants.

A series of intertypic (type 3/type 1) poliovirus recombinants was obtained whose crossover sites were expected to be located in the middle of the viral genome, between the loci encoding type-specific antigenic properties, on the 5' side, and an altered sensitivity to guanidine, on the 3' side. The primary structures of the crossover regions in the genomes of these recombinants were determined by the primer extension method. The length of the crossover sites (the uninterrupted sequences shared by the recombinant and both parental genomes that are flanked, in the recombinant RNAs, by two heterotypic segments) varied between 2 and 32 nucleotides, but the majority of the sites were 5 nucleotides long or shorter. The crossover sites were nonrandomly distributed over the presumably available genome region: only a single such site was found within the gene for polypeptide 2A, whereas an apparent clustering of the crossover sites was encountered in other genomic segments. When the crossover sites were superimposed on a model of the secondary structure of the relevant region of the viral RNA molecule, a pattern consistent with the previously proposed mechanism of poliovirus recombination (L.I. Romanova, V.M. Blinov, E.A. Tolskaya, E.G. Viktorova, M.S. Kolesnikova, E.I. Guseva, and V.I. Agol (1986) Virology 155, 202-213) was observed. It is suggested that the nonrandom distribution of the crossover sites in the genomes of intertypic poliovirus recombinants was due to two factors: the existence of preferred sites for recombination, and selection against recombinants with a lowered level of viability.

Animals↗
PubMed1988

Replication of the murine cytomegalovirus genome: structure and role of the termini in the generation and cleavage of concatenates.

Following infection with murine cytomegalovirus (MCMV), the termini of the linear double-stranded DNA genome fuse to form circular or concatemeric forms which serve as replicative intermediates. To investigate the mechanisms involved in the generation and cleavage of the intracellular concatenates, we have used restriction endonuclease mapping and nucleotide sequence analyses to characterize the structure of the virion DNA termini and intracellular end-to-end fusion fragment. Four each of the cloned EcoRI X and EcoRI c terminal fragments were sequenced. All of the EcoRI X clones and three of the EcoRI c clones contained a 30-base-pair (bp) sequence which was directly repeated at the ends of the MCMV genome. The terminal sequence of the fourth EcoRI c clone began directly after the 30-bp direct repeat. The four EcoRI X clones also had minor length heterogeneity, differing in the number of GC bp at the terminus. Five fusion fragments were sequenced. Three of the fusion fragments contained both direct repeats, while two fusion fragments lacked one entire direct repeat. Direct analyses of the virion DNA and intracellular fusion fragments revealed that the clones accurately reflected the naturally occurring populations and that the relative proportion of fusion fragments containing only one direct repeat increased as the infection progressed. The data suggest that fusion of the termini arises by end-to-end ligation. We also show that adjacent to the MCMV termini are sequences highly conserved among the herpesviruses, and we discuss their potential role in the maturation of the viral genome.

Cloning, Molecular↗
PubMed1989

Genetic variation and microevolution of dengue 2 virus in Southeast Asia.

Dengue 2 (DEN 2) virus strains collected from dengue hemorrhagic fever (DHF) patients and Aedes aegypti mosquitoes in Thailand, Burma, and Vietnam over a 25-year period have been analyzed by computer assisted T1-RNase-resistant oligonucleotide fingerprinting. Fifty-seven DEN 2 virus strains of the Thailand topotype were separated into four major clusters by phylogenetic analysis of 97 unique oligonucleotides identified in a common well-resolved region of the fingerprints. Similarities in the 57 fingerprints indicated that DEN 2 virus of a single, continually evolving genetic population has been involved in endemic transmission of the disease. Virus isolates from DHF cases and mosquitoes are genetically very similar, indicating that different genetic topotypes are not selectively the cause of severe DEN disease in Thailand. Microevolution of the DEN 2 virus genome from 1962-1986 was gradual with detectable changes in the pattern of oligonucleotides through time. Segregation of the DEN 2 virus fingerprints into the three decades (1960s, 1970s, and 1980s) revealed the rate of genetic change to be one consensus oligonucleotide per year. Based on average association coefficient (Sab) values between the consensus fingerprints for each decade, the similarity between the consensus fingerprints decreased by 1.4% per year. Genetic variation during each of the three decades was found to be essentially the same (0.866 +/- 0.053). Constancy in the microevolutionary rate and genetic variability suggests that a balance of genetic drift and natural selection acting on the viral population did not significantly change throughout the 25-year period.

Aedes↗
PubMed1989

Genetic heterogeneity of the RNA genome population of the plant virus U5-TMV.

The genetic heterogeneity in a population of the U5 strain of tobacco mosaic virus (U5-TMV) was studied. The T1 fingerprint characterizing a cloned population did not vary after a new cloning step in the local lesion host Nicotiana tabacum Xanthi-nc, nor during four series of 20 passages in the systemic host N. tabacum Samsum. No heterogeneity was observed among 10 clones derived from the cloned populations, while 1 of 18 clones derived from a 20-fold passaged population differed from the rest in 1 of 55 oligonucleotides. A higher heterogeneity was found in an uncloned field isolate in which 2 of 10 clones differed in type in 1 and 2 oligonucleotides, respectively. These data agree with those reported for bacterial and animal RNA viruses and are compatible with the quasi-species model for RNA populations. On the other hand, the intrapopulational heterogeneities found for U5-TMV are considerably smaller than those reported for other RNA viruses, our data showing a high genetic stability for U5-TMV.

Cloning, Molecular↗
PubMed1990

Transcription of the human cytomegalovirus glycoprotein gene family in the short unique component of the viral genome.

The HXLF (HindIII X left reading frame) genes are a group of six open reading frames (ORFs) within the short unique component of the human cytomegalovirus genome. Two or more ORFs of the HXLF gene family code for components of the virion envelope glycoprotein complex designated gcII. Transcription of the various HXLF genes in infected cells was analyzed using specific DNA, RNA, and synthetic oligonucleotide probes. Transcription from each of the six HXLF genes was analyzed at immediate early, early, and late times after infection. Even though an enhancer element in six copies is downstream of HXLF6, immediate early cytoplasmic RNAs from the HXLF genes was not detectable. In contrast, early and late cytoplasmic RNAs were detectable for all six HXLF genes. The HXLF1 and 2 genes and the HXLF3 and 4 genes are transcribed as bicistronic mRNAs of 1.6 and 1.7 kb, respectively. The HXLF5 and 6 genes are transcribed as monocistronic mRNAs. In the HXLF4, 5, and 6 transcription units cytoplasmic viral RNAs were detected from both strands. Bidirectional transcription upstream of the enhancer element occurs at only early and late times after infection. The significance of bicistronic mRNA to the formation of the glycoprotein complex gcII requires further investigation.

Blotting, Northern↗
PubMed1990

Pathogenicity of BALB/c-derived N-tropic murine leukemia viruses.

N-tropic murine leukemia viruses have been observed in connection with radiation-induced osteosarcomagenesis in BALB/c mice. We have investigated the bone disease-inducing potential of molecularly cloned, BALB/c-derived N-tropic viruses in the random-bred NMRI mouse strain. The germ-line virus and an exogenous virus isolate were found to induce high incidences of osteopetrosis and lymphomas and a lower incidence of osteomas. Two viruses derived from somatically acquired proviruses of independent radiation-induced osteosarcomas induced lower incidence of osteopetrosis and lymphomas. Nucleotide sequence analysis of the long terminal repeat regions and RNase T1 fingerprint analysis revealed only few differences between the isolates. The possible involvement of N-tropic murine leukemia viruses in radiation-induced osteosarcomagenesis in the BALB/c mouse strain is discussed.

Animals↗
PubMed1990

The Sindbis virus 6K protein can be detected in virions and is acylated with fatty acids.

A small hydrophobic polypeptide is encoded within the genome of the alphaviruses by a set of 165 nucleotides which map between the sequences for the two virus glycoproteins. This polypeptide has been referred to as 6K and was previously found on membranes in virus-infected cells. We report here that this protein is heavily acylated with long chain fatty acids covalently attached in hydroxylamine-sensitive ester bonds and that the 6K protein can be detected in purified preparations of virions. A polyclonal rabbit serum, raised against a peptide which contained the 16 amino acids at the amino-terminus of the 6K protein, was used to identify the 6K protein in infected cells and virions. This antibody also precipitated a 4K protein which was present in Sindbis virus-infected cells but not in virions. This latter protein was shown to be an underacylated form of the 6K protein and infected cells contained about twice as much 4K as 6K. In the cell there was close to a 1:1 stoichiometry between the 4K + 6K proteins and the virus glycoproteins E1, p62, and E2, but in virions the ratio of 6K to E1 + E2 ranged from 0.08 to 0.12.

Acylation↗