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Biomedical subjects

M Woodworth-Gutai

Publications and source records attributed to M Woodworth-Gutai.

16 recordsLinked to original sources
PubMed1986

Evolutionarily selected replication origins: functional aspects and structural organization.

A selective replicative pressure occurs during the evolution of simian virus 40 variants. When the replication origin is duplicated as an inverted repeat, there is a dramatic enhancement of replication. Having regulatory sequences located between the inverted repeat of ori magnifies their enhancing effect on replication. A passage 20 variant and a passage 45 variant containing three pairs of an inverted repeat of ori replicated more efficiently than a passage 13 variant containing nine copies of ori arranged in tandem. A 69-base-pair cellular sequence inserted between inverted repeats of ori of both passage 40 and 45 variants enhanced simian virus 40 DNA replication. Differences in replication efficiencies became greater as the total number of replicating species was increased in the transfection mixture, under conditions where T antigen is limiting. In a competitive environment, sequences flanking the replication origin may be inhibitory to replication.

Animals↗
PubMed1986

Simian virus 40 DNA replication: functional organization of regulatory elements.

The efficiency of simian virus 40 (SV40) DNA replication is dependent on the structural organization of the regulatory region. The enhancing effect of the G + C-rich 21-base-pair (bp) repeats on SV40 DNA replication is position and dose dependent and to some extent orientation dependent. The inverted orientation is about 50% as effective as the normal orientation of the 21-bp repeat region. Movement of the 21-bp repeat region 180 or 370 bp upstream of the ori sequence abolishes its enhancing effect, whereas no replication is detected if the 21-bp repeat region is placed downstream of the ori sequence. The dose-dependent enhancement of the 21-bp repeat of SV40 DNA replication as first described in single transfection by Bergsma et al. (D. J. Bergsma, D. M. Olive, S. W. Hartzell, and K. N. Subramanian, Proc. Natl. Acad. Sci. USA 79:381-385, 1982) is dramatically amplified in mixed transfection. In the presence of the 21-bp repeat region, the 72-bp repeat region can enhance SV40 DNA replication. In the presence of the 21-bp repeats and a competitive environment, the 72-bp repeat region exhibits a cis-acting inhibitory effect on SV40 DNA replication.

Animals↗
PubMed1984

Subfamilies of the mouse major urinary protein (MUP) multi-gene family: sequence analysis of cDNA clones and differential regulation in the liver.

The mouse major urinary proteins (MUPs) are the products of a multi-gene family of 30-35 genes whose members exhibit diverse tissue specific, developmental, and hormonal controls. Three cDNA clones corresponding to liver MUP mRNAs have been sequenced. Two of the clones (p499, C57BL/6 and p1057, BALB/c) share strong homology whereas a third clone (p199, C57BL/6) has diverged considerably from the others at the nucleic acid (85% homology) and protein (68% homology) levels. The 5' regions of p499 and p199 which show the most sequence divergence were subcloned and shown to hybridize to different liver MUP mRNAs. The p499-5' sequence was expressed in all MUP expressing tissues (liver, lachrymal, submaxillary and mammary) whereas the p199-5' sequence was expressed primarily in the liver and lachrymal. Analysis of liver RNA from mice in different endocrine states indicates that the p499-5' sequence is strongly regulated by thyroxine administration whereas the p199-5' sequence is not. Both sequences appear to be regulated by growth hormone and testosterone. Southern blot analysis of mouse genomic DNA indicates that there are multiple genes homologous to each sequence.

Animals↗
PubMed1984

Use of a cDNA recombinant for the gamma-subunit of mouse nerve growth factor to localize members of this multigene family near the TAM-1 locus on chromosome 7.

The gamma-subunit of mouse 7S nerve growth factor (gamma-NGF) is a member of a family of closely related serine proteases that includes kallikreins and tamases. We have isolated from a DBA/2J male submaxillary gland cDNA library a clone, pSM676, which codes for gamma-NGF. Sequence analysis of the clone shows that it codes for the C-terminal 138 amino acids of the protein plus 23 bases of the 3'-nontranslated portion of the message. The predicted amino acid sequence agrees with that determined by Thomas et al. (1) for the gamma-subunit of nerve growth factor from Swiss Webster mice except for the single, conservative substitution of glutamate for aspartate at amino acid 175. When used as a probe for Southern blot analysis, pSM676 hybridizes to at least twelve fragments of restricted mouse genomic DNA which correspond to several different serine protease genes. Using mouse-hamster hybrid cell lines and recombinant inbred strains of mice, we have demonstrated that all of the genes which show homology to pSM676 are located on mouse chromosome 7, clustered at or near the Tam-1 locus.

Animals↗
PubMed1984

Most highly repeated dispersed DNA families in the mouse genome.

The construction of a small library of mouse repetitive DNA has been previously reported (Pietras et al., Nucleic Acids Res. 11:6965-6983, 1983). Here we report that the 35 plasmids in this library corresponding to highly repeated (greater than 30,000 copies per genome) dispersed DNA sequences can be grouped into no more than 5 distinct families. These families together comprise 8 to 10% of the mouse genome. They include the previously described small elements B1, B2, and R and the large MIF-1 element. Twelve of the 35 clones contain evolutionarily conserved (EC) sequences. One EC clone in our library mostly consists of alternating dCdT residues; another consists of tandem repeats of the sequence CCTCT. The majority of B1s and B2s in the genome appear to be homogeneous, whereas R sequences, ECs, and MIF-1s are heterogeneous. Two earlier reports showed highly repeated mammalian DNA sequences in the herpesvirus genome (Peden et al., Cell 31:71-80, 1982; Puga et al., Cell 31:81-87, 1982). We show that sequences homologous to our EC clones are present in the herpesvirus genome, although these polypyrimidine stretches are not detected in poxvirus, adenovirus, and simian virus 40 genomes. We detect transcripts containing homology to all of these sequences in a nuclear transcription assay. Also, we show that small, polyadenylated RNA molecules homologous to B2 sequences are expressed in undifferentiated embryonal carcinoma cells but not in their differentiated derivatives. The significance of these findings is discussed.

Animals↗
PubMed1983

Recombination in simian virus 40-infected cells. Structure of naturally arising variants ev-2114, ev-2102, and ev-1110.

The complete nucleotide sequence has been determined for three newly cloned evolutionary variants from two different independently generated evolutionary series (1100 and 2100 series) of simian virus 40 (SV40). These naturally arising variants, designated ev-1110, ev-2102, and ev-2114, were isolated after five high multiplicity serial passages. The structure of the variants consists of a monomeric unit tandemly repeated four times (ev-2102 and ev-2114) or six times (ev-1110) in the variant genome; the variants have four or six copies, respectively, of the viral origin signal for DNA replication. The DNA content in the three variants is vastly different in that the genome of variant ev-2114 contains only rearranged viral sequences, while variant ev-2102 contains a substitution with monkey DNA sequences consisting of a nearly complete dimeric unit of Alu family sequences as well as less repetitive sequences and variant ev-1110 contains monkey DNA sequences derived solely from repetitive alpha-component DNA. Recombination events, cellular sequences, and structural features of these and other naturally arising SV40 variants are compared.

Base Sequence↗
PubMed1983

Construction of a small Mus musculus repetitive DNA library: identification of a new satellite sequence in Mus musculus.

We report the construction of a small library of recombinant plasmids containing Mus musculus repetitive DNA inserts. The repetitive cloned fraction was derived from denatured genomic DNA by reassociation to a Cot value at which repetitive, but not unique, sequences have reannealed followed by exhaustive S1 nuclease treatment to degrade single stranded DNA. Initial characterizations of this library by colony filter hybridizations have led to the identification of a previously undetected M. musculus minor satellite as well as to clones containing M. musculus major satellite sequences. This new satellite is repeated 10-20 times less than the major satellite in the M. musculus genome. It has a repeat length of 130 nucleotides compared with the M. musculus major satellite with a repeat length of 234 nucleotides. Sequence analysis of the minor satellite has shown that it has a 29 base pair region with extensive homology to one of the major satellite repeating subunits. We also show by in situ hybridization that this minor satellite sequence is located at the centromeres and possibly the arms of at least half the M musculus chromosomes. Sequences related to the minor satellite have been found in the DNA of a related Mus species, Mus spretus, and may represent the major satellite of that species.

Animals↗
PubMed1983

Naturally arising recombinants that are missing portions of the simian virus 40 regulatory region.

When simian virus 40 (SV40) is serially passaged at high multiplicity, a heterogeneous collection of naturally arising variants is generated. Those which are the most abundant presumably have a selective replicative advantage over other defective and wild-type helper SV40s. Two such naturally arising host-substituted variants of SV40 have been characterized in terms of complete nucleotide sequence determination. Evolutionary variant ev-1101 (previously isolated by Lee et al., Virology 66:53-69, 1975) is from undiluted serial passage 13, whereas ev-2101 is newly isolated from undiluted serial passage 6 of an independently-derived evolutionary series. Both variants contain a five-times tandemly repeated segment of DNA consisting of viral Hin C and Hin A sequences that have recombined with a segment of host DNA that is not highly reiterated in the monkey genome. The monkey segment differs in the two variants as does the size of the viral segment retained. In two additional host-substituted variants, ev-1102 (previously isolated from serial passage 20 by Brockman et al., Virology 54:384-397, 1973) and ev-1108 (newly isolated from serial passage 40), the SV40 sequences derived from the replication origin are present as inverted repetitions. The inverted repeat regions of these two variants have been analyzed at the nucleotide sequence level and are compared with SV40 variant ev-1104 from passage 45 (previously characterized by Gutai and Nathans, J. Mol. Biol. 126:259-274, 1978). The viral segment containing the regulatory signals for replication and viral gene expression is considerably shortened in later serial passages as demonstrated by these five variants. It is of interest that the variants presumably arose due to their enhanced replication efficiency, yet are missing some of the sequence elements implicated in the regulation of replication. Furthermore, a comparison of the structure of the replication origin regions indicates that additional changes occur in the SV40 regulatory region with continued undiluted serial passage.

Base Sequence↗
PubMed1983

Organization of rDNA spacer fragment variants among human acrocentric chromosomes in somatic cell hybrids.

Although the human rDNA gene family is organized in clusters of tandem repeats on five pairs of acrocentric chromosomes, all rDNA genes have undergone a "concerted" evolution resulting in a homogeneous population of genes. Two steps are necessary for a variant to spread to all rDNA genes: dissemination of the variant to all genes in a cluster; and exchange of rDNA genes between nonhomologous chromosomes. To study the organization of rDNA genes, a restriction fragment length polymorphism in the spacer region adjacent to the 28S gene was examined in somatic cell hybrids in which individual human acrocentric chromosomes could be isolated. Human DNA cut with BamHI and analyzed by Southern hybridization yields two to four major bands that hybridize to a 32P-labeled cloned fragment of the 28S gene. Hybrids containing single human acrocentric chromosomes do not recapitulate the parental patterns, but frequently have only one of the parental bands. The data suggest that the quantitative distribution of spacer length variants differs among the human acrocentric chromosomes in hybrids. The frequently observed homogeneity of the rDNA variants on individual acrocentric chromosomes in hybrid cells may reflect the individual rDNA clusters in the parental cell or may be a result of unequal crossing over in the hybrid cell.

Animals↗
PubMed1982

Deletion mutants which affect the nuclease-sensitive site in simian virus 40 chromatin.

A short segment of simian virus 40 (SV40) chromatin on the late side of the origin of replication is hypersensitive to nuclease cleavage. The role of DNA sequence information in this nuclease-sensitive feature was examined by constructing deletion mutations in this region. Deletions were introduced into the inserted segment of in(Or)-1411 (a viable, partially duplicated variant of SV40), and nuclease sensitivity of the inserted segment was compared with that of the unaltered sequences in their normal location in the viral genome. Extended deletions (118 to 161 base pairs) essentially abolished nuclease sensitivity within the inserted segment. Shorter deletions (21 to 52 base pairs) at separate locations retained the nuclease-sensitive feature. In some short-deletion mutants nuclease susceptibility was substantially reduced. We conclude that more than one genetic element in this region contributes to the organization of the nuclease-sensitive feature and that the SV40 72-base repeat is not, in itself, sufficient signal for this feature.

Animals↗
PubMed1977

Ultraviolet light irradiation of PM2 superhelical DNA.

Superhelical PM2 DNA can be photochemically modified by u.v. irradiation. The variation of S20,w with dose shows the following characteristics. There is a linear increase from 28 to 31s produced by a low dose of u.v. irradiation (4,000 ergs/mm2). A plateau in S20,w occurs between 4,000 and 10,000 ergs/mm2. The S20,w then increases when irradiation is increased to 56,000 ergs/mm2. Thymine dimers are introduced proportional to dose throughtout the range of exposure to u.v. light. Sedimentation velocity-dye titrations reveal anomolous behavior, i.e. apparent increases in superhelix density (sigma). However, the dye-buoyant density procedure showed no change in sigma under the same conditions. The most satisfactory model for the data is preferential photochemical modification of premelted (possibly hairpin) sites as a greater rate than the introduction of photoproducts into duplex sites. The origin of the anomoly in the sedimentation velocity dye titrations is still unclear.

Bacteriophages↗
PubMed1976

Introduction of interrupted secondary structure in supercoiled DNA as a function of superhelix density: consideration of hairpin structures in superhelical DNA.

PM2 DNA was prepared with different superhelical densities (sigma) in order to examine the relationship betweenn supercoiling and the occurrence of a region(s) of unpaired bases in this DNA. A previous study showed that CH3HgOH reacts with native superhelical PM2 DNA more rapidly than the nicked form II. This evaluation of binding, monitored through the change of sedimentation velocity, was repeated on PM2 DNA I with different superhelical densities. Early binding is detected by an increase in sedimentation velocity and occurs with molecules with sigma' values betwee -0.025 and -0.037. The conversion of form I to form II with the single-strand-specific endonuclease from Neurospora crassa also occurs above a sigma value of -0.025. This data strongly supports the view that supercoiling produces interrupted secondary structure. The question whether the interrupted regions remain single stranded in character or form small intrastrand hairpin regions is considered by examining which model best fits the CH3HgOH- induced sedimentation velocity changes and the standard sedimentation velocity versus the superhelical density curve for the in vitro made DNAs. The hairpin model offers the most satisfactory explanations for all the results of this and previous studies.

Bacteriophages↗
PubMed1972

New deoxyribonuclease activity after bacteriophage P22 infection.

Extracts from P22-infected and uninfected cultures of Salmonella typhimurium were subjected to deoxyribonucleic acid (DNA)-cellulose and diethylaminoethyl-cellulose chromatography. Comparison of the elution patterns revealed that in infected cells there is a decrease in the amount of nuclease activity specific for denatured DNA and an increase in the amount of nuclease activity specific for native DNA. The latter activity was shown to differ from a similar host enzyme in Mg(2+), Mn(2+), and pH optima. This new activity is not found after infection of a lysogen with a nonvirulent phage or after infection under nonpermissive conditions with P22ts25.1 (a mutant in gene 25 that carries out no known functions other than adsorption and injection) and thus appears to be specified by the phage genome.

Ammonium Sulfate↗
PubMed1972

Inhibitory effect of bacteriophage P22 infection on host cell deoxyribonuclease activity.

Infection of Salmonella typhimurium with phage P22 causes a decrease in the activity of host deoxyribonuclease which degrades single-stranded deoxyribonucleic acid (DNA). This decrease is reversed when the infecting phage is P22c(+); it is not reversed if the infecting phage kills the cell. The decrease does not occur in infections with P22ts25.1 (which only adsorbs and injects DNA) or in infections of a lysogen by a nonvirulent phage. It does occur, however, after infections with other phages which are blocked in phage DNA synthesis. Inhibiting protein synthesis with chloramphenicol does not in itself cause the decrease in uninfected cells, but it does prevent infected cells from showing this effect.

Chloramphenicol↗