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PubMed1982

Binding of the origin of replication of Escherichia coli to the outer membrane.

The replication origin of the Escherichia coli chromosome binds with high affinity to outer membrane preparations. This binding requires a 460 bp stretch of origin DNA between positions -40 and 420 of the oriC map. Specific binding can be detected by the use of a membrane filter retention assay in the presence of excess calf thymus DNA. This binding is enhanced by divalent cations and takes place specifically at a few (0.7-3.0) membrane sites per cell. The apparent affinity of origin DNA for membranes is enhanced by two peptides, (55 kilodaltons (kd) and 75 kd), which remain attached to the DNA through treatment with 5.5 M cesium chloride.

Bacterial Proteins↗
PubMed1996

Mapping of replication initiation sites in the mouse ribosomal gene cluster.

We have used nascent strand determination analysis to map start sites of DNA replication in the mouse ribosomal gene cluster in which individual copies of the ribosomal genes are separated by intergenic spacer regions. One origin of bidirectional replication (OBR) was localized within a 3 kb region centered about 1.6 kb upstream of the rDNA transcription start site. At least one additional initiation site is situated near the 3' end of the transcription unit. Adjacent to the OBR at the transcription start site are located two amplification-promoting sequences, i.e., APS1 and APS2. Nuclease-hypersensitive sites were identified in both of the two APSs as well as in the OBR region, thus indicating that these sequences have an altered chromatin structure. In the OBR an intrinsically bent region, a purine-rich element and other prospective initiation zone components are found.

3T3 Cells↗
PubMed1980

Viruses isolated from cells persistently infected with vesicular stomatitis virus show altered interactions with defective interfering particles.

Virus mutants isolated from persistent infections of vesicular stomatitis virus in BHK-21 cells were much less susceptible to interference mediated by the defective interfering particle used to establish the persistent infection. This mutational change occurred as early as 34 days in the persistent infection and continued for over 5 years. The earliest variants showed no oligonucleotide map changes and no difference in the temperature-sensitive phenotype from the original virus, but the later variants exhibited extensive map changes. These results suggest a possible role for defective interfering particles in the selection of the mutants.

Animals↗
PubMed1990

Analysis of the rearrangements associated with carcinogen-induced activation of the hamster thymidine kinase gene.

We have previously shown that chemical carcinogen treatment of RJK92 hamster cells activates the quiescent thymidine kinase (TK) gene and that 20% of the TK+ variants have a rearrangement in the region 5' to the TK gene (Barr et al. (1986) Mol. Cell. Biol. 6, 3023-3033). After cloning the wild type 5' region to obtain detailed mapping data and hybridization probes, we localized the rearrangement breakpoints by Southern blot analysis to a 1.5 kb region 6 kb 5' to the origin of transcription. This analysis also demonstrated that the rearrangements consist at least partly of a deletion of wild type sequences 5' to this breakpoint region. The region near the transcription origin in the rearranged TK genes has a DNase I-sensitive chromatin conformation and a DNase I hypersensitive site as well as the previously described domain of demethylation (Ibid.). Though this domain of demethylation extends into the breakpoint region, the rearranged region is not associated with DNase I sensitivity nor hypersensitive sites. The rearrangement also does not detectably alter the growth-related regulation of TK activity in these cells.

Animals↗
PubMed1996

Initial organization of neurons and tracts in the embryonic mouse fore- and midbrain.

We investigated the potential role of rostral-caudal and dorsal-ventral subdivisions of the early rostral brain by relating these subdivisions to the early patterning of neuron cell bodies and their axon projections. The earliest neurons were mapped using the lipophilic axon tracers diI and diO on embryos fixed on embryonic days 9.5-10.5 (E9.5-E10.5); neuromeric boundaries were marked by diO. The tracts were small in number, were organized orthogonally (2 dorsal-ventral and 4 rostral-caudal), and originated from groups of cell bodies which we term "sources." Two parallel longitudinal axon systems, one dorsal (the tract of the postoptic commissure and the mesencephalic tract of the trigeminal nerve) and one ventral (the mammillotegmental tract and the medial longitudinal fasciculus), projected caudally from the prosencephalon into the rhombencephalon. We argue that the dorsal longitudinal pathway marked the boundary between the alar and basal plates along the entire neuraxis. The dorsal-ventral axons coursed circumferentially and either crossed the midline (forming the posterior and ventral tegmental commissures) or turned caudally without crossing the midline. The dorsal-ventral axons were not generally restricted to the interneuromeric boundaries, as others have suggested. Earlier, all neighboring neurons projected their axons together; later, nearby neurons projected into different pathways. Some tracts originated in single neuromeres, while other tracts had origins in two or more neuromeres. The dorsal longitudinal axons altered course at several of the borders, but the ventral longitudinal axons did not. In summary, the early subdivisions appeared to influence some, but not all, aspects of tract formation.

Animals↗
PubMed1994

Identification of novel transcripts complementary to the Marek's disease virus homologue of the ICP4 gene of herpes simplex virus.

Libraries of cDNA were generated from polyadenylated RNAs derived from Marek's disease virus (MDV)-transformed cell lines by directional cloning of oligo-(dT)-primed cDNAs in lambda gt22A. Analysis of the libraries for viral sequences showed that a number of cDNA clones originated from transcripts mapping in the BamHI A region of the MDV genome. Sequencing and fine mapping of these cDNAs suggested that the RNA transcripts expressed from this region were either in the sense or antisense direction with respect to the MDV homologue of the ICP4 gene of herpes simplex virus. The longest cDNA clone from antisense transcripts was 2756 bp long and partially overlapped the 5' end of the coding region of the ICP4 gene. The cDNA clone contained at least four introns, shown by comparison of its sequence with the sequence of the ICP4 gene. The presence of introns was confirmed by PCR analysis. All the introns have the consensus splice donor and acceptor signals at their 5' and 3' ends respectively. Northern blot analysis showed that the ICP4 gene homologue of MDV was abundantly transcribed only in lytically infected fibroblasts, whereas transcripts complementary to the ICP4 gene were the major transcripts in MDV-transformed cell lines and lymphomas. The transcripts complementary to ICP4 consist of two major RNA species approximately 15 kb and 1.32 kb long. The results suggest that there might be an inverse relationship between the abundance of ICP4 transcripts and their complementary transcripts in MDV-infected and transformed cells.

Amino Acid Sequence↗
PubMed2001

A dominant modifier of transgene methylation is mapped by QTL analysis to mouse chromosome 13.

The single-copy hepatitis B virus transgene in the E36 transgenic mouse strain undergoes methylation changes in a parent-of-origin, tissue, and strain-specific fashion. In a C57BL/6 background, the paternally transmitted transgene is methylated in 30% of cells, whereas it is methylated in more than 80% of cells in (BALB/c x C57BL/6) F1 mice. We established previously that several genetic factors were likely to contribute to the transgene methylation profile, some with demethylating and some with de novo methylating activities. Using quantitative trait loci (QTL) mapping, we have now localized one major modifier locus on chromosome 13 (Mod13), which explains a 30% increase in the methylation level of this transgene with no effect on the flanking endogenous sequences. No other QTL could be identified, except for a demethylating activity of low significance located on chromosome 12. Recombinant inbred mice containing a BALB/c allele of Mod13 were then used to show that the presence of Mod13 is sufficient to induce de novo methylation. A segregation between de novo methylation and repression of transgene expression was uncovered, suggesting that this genetic system is also useful for the identification of factors that interpret methylation patterns in the genome.

Animals↗
PubMed2000

Mouse scrapie responsive gene 1 (Scrg1): genomic organization, physical linkage to sap30, genetic mapping on chromosome 8, and expression in neuronal primary cell cultures.

We have previously reported a transcript of a novel mouse gene (Scrg1) with increased expression in transmissible spongiform encephalopathies and the cloning of the human mRNA analogue. In this paper, we present the genomic organization of the mouse and human SCRG1 loci, which exhibit a high degree of conservation. The genes are composed of three exons; the two downstream exons contain the protein coding region. The mouse gene is expressed in brain tissue essentially as a 0.7-kb message but also as a minor 2.6-kb mRNA. We have sequenced 20 kb of DNA at the mouse Scrg1 locus and found that the longer transcript is the prolongation of the 0.7-kb mRNA to a polyadenylation site located about 2 kb further downstream. Sequencing revealed that the mouse Scrg1 gene is physically linked to Sap30, a gene that encodes a protein of the histone deacetylase complex, and genetic linkage mapping assigned the localization of Scrg1 to chromosome 8 between Ant1 and Hmg2. Northern blot analysis showed that Scrg1 is under strict developmental control in mouse embryo and is expressed by cells of neuronal origin in vitro. Comparison of the rat, mouse, and human SCRG1 proteins identified a box of 35 identical contiguous amino acids and a characteristic cysteine distribution pattern defining a new protein signature.

Amino Acid Sequence↗
PubMed1982

Genetic mapping of mutation in Escherichia coli leading to a temperature-sensitive RNase D.

In order to determine the metabolic role of RNase D in Escherichia coli, we have attempted to isolate strains deficient in this enzyme. One strain containing a temperature-sensitive RNase D was found among a heavily mutagenized stock of strain temperature-sensitive for growth. Genetic mapping of the mutation responsible for the altered RNAse D enabled us to define the rnd locus, at 39.5-40.0 min on the E. coli map, which apparently specifies the RNase D structural gene. Using a Tn10 insertion near the rnd locus, we constructed isogenic strains containing RNase D and Rnase II mutations, alone or in combination. Although the original mutant isolate displayed temperature-sensitive growth. no growth phenotype was associated with the rnd mutation in wild type background, possibly because a substantial amount of RNase D remained in cells grown at 45 degrees C. However, elucidation of the map position of the rnd locus should prove useful for the isolation of other mutant strains with lower levels of RNase D.

Alleles↗
PubMed1999

Eukaryotic DNA replication.

One of the fundamental characteristics of life is the ability of an entity to reproduce itself, which stems from the ability of the DNA molecule to replicate itself. The initiation step of DNA replication, where control over the timing and frequency of replication is exerted, is poorly understood in eukaryotes in general, and in mammalian cells in particular. The cis-acting DNA element defining the position and providing control over initiation is the replication origin. The activation of replication origins seems to be dependent on the presence of both a particular sequence and of structural determinants. In the past few years, the development of new methods for identification and mapping of origins of DNA replication has allowed some understanding of the fundamental elements that control the replication process. This review summarizes some of the major findings of this century, regarding the mechanism of DNA replication, emphasizing what is known about the replication of mammalian DNA. J. Cell. Biochem. Suppls. 32/33:1-14, 1999.

Animals↗
PubMedDate not supplied

Mapping and characterizing a new DNA replication mutant in Saccharomyces cerevisiae.

A detailed characterization of the mak1-3 mutation of Saccharomyces cerevisiae has been made possible by modifying its genetic background. The mak1-3 mutation, which confers temperature sensitivity for growth, was originally identified as one of four mak1 mutations (Wickner and Leibowitz, 1976). Mak1-1, 1-2 and 1-4 mutants are deficient in DNA topoisomerase I activity and thus have been renamed 'top1' (Thrash et al., 1984). Studies presented here show that the map position of MAK1-3 on chromosome XVI distinguishes it from TOP1 which maps on chromosome XV (Wickner and Leibowitz, 1976). An investigation of in vivo macromolecular synthesis in the mak1-3 mutant shows that it is deficient in DNA replication at the restrictive temperature. Experiments in which DNA synthesis was measured in synchronized cell populations indicate that the mak1-3 mutant is deficient in the initiation step of DNA synthesis. Furthermore, crude extracts from the mak1-3 mutant cells support temperature-sensitive in vitro DNA synthesis on yeast chromosomal DNA replication origin containing plasmid pARS1, suggesting that the MAK1 gene product is directly required for in vitro DNA replication. The conclusion that mak1-3 is a newly identified DNA replication mutation is based on the observations that it (1) complements all DNA synthesis mutants examined, (2) maps to a previously undetected chromosomal location and (3) has a distinct terminal morphology. In light of these distinctions and of the role mak1-3 plays in DNA replication, it has been renamed 'dna1'.

Chromosome Mapping↗
PubMed1990

Structure and molecular analysis of RGR1, a gene required for glucose repression of Saccharomyces cerevisiae.

An RGR1 gene product is required to repress expression of glucose-regulated genes in Saccharomyces cerevisiae. The abnormal morphology of rgr1 cells was studied. Scanning and transmission electron microscopic observations revealed that the cell wall of the daughter cell remained attached to that of mother cell. We cloned the RGR1 gene by complementation and showed that the cloned DNA was tightly linked to the chromosomal RGR1 locus. The cloned RGR1 gene suppressed all of the phenotypes caused by the mutation and encoded a 3.6-kilobase poly(A)+ RNA. The RGR1 gene is located on chromosome XII, as determined by pulsed-field gel electrophoresis, and we mapped rgr1 between gal2 and pep3 by genetic analysis. rgr1 was shown to be a new locus. We also determined the nucleotide sequence of RGR1, which was predicted to encode a 123-kilodalton protein. The null mutation resulted in lethality, indicating that the RGR1 gene is essential for growth. On the other hand, a carboxy-terminal deletion of the gene caused phenotypes similar to but more severe than those caused by the original mutation. The amount of reserve carbohydrates was reduced in rgr1 cells. Possible functions of the RGR1 product are discussed.

Alleles↗
PubMed1989

Projection from the superficial layers of the tectum to the pretectal complex in the cat.

The projection from the superficial layers of the tectum to the pretectal complex in the cat was examined using the retrograde transport of wheat germ agglutinin-horseradish peroxidase. Restricted injections were made into different parts of the pretectum. Neurons in the superior colliculus were found to be arranged in a mediolateral array that corresponds to the rostromedial to caudolateral array of their axon projections to the nucleus of the optic tract and posterior pretectal nucleus. These results suggest that similar parts of the retinotopic maps present in the pretectum and superior colliculus are connected. The labeled cells in the superior colliculus were located within the deep part of stratum griseum superficiale and the superficial part of stratum opticum, and were composed of multipolar cells, vertical fusiform cells and horizontal cells. We conclude from this study that the cells of origin in the superior colliculus originate in the same region to which the contralateral retinal Y-cells project and that have also the morphological diversity, as do other tecto-thalamic neurons.

Animals↗
PubMed1990

Avian endogenous provirus (ev-3) env gene sequencing: implication for pathogenic retrovirus origination.

The avian endogenous env gene product blocks the surface receptor and, as a result, cells become immune to related exogenous retroviruses. On the other hand, the same sequence can be included in the pathogenic retrovirus genome, as shown by oligonucleotide mapping. However, since the complete env gene sequence was not known, the comparison of genomic nucleotide sequences was not possible. Therefore an avian endogenous provirus with an intact env gene was cloned from a chicken gene bank and the regions coding for the C terminus of the gp85 and gp37 proteins were sequenced. Comparison of this sequence with those of other retroviruses proved that one of the pathogenic viruses associated with osteopetrosis is a cross between avian endogenous virus and Rous sarcoma virus. Retroviruses and, especially, endogenous retroviruses are traditionally of the most developed models of viral carcinogenesis. Many endogenous retroviruses are implicated in neoplastic transformation of the cell. For instance, endogenous mouse mammary tumor virus of some inbred lines appears to be the only causative agent in these mammary cancers. Other even nonpathogenic murine endogenous retroviruses are involved in the origination of MCF-type recombinant acute leukosis viruses. Some endogenous retroviruses are implicated in the transduction or activation of cellular protooncogenes. Our interest in endogenous viruses is based on their ability to make cells resistant to exogenous retroviruses. Expression of their major envelope glycoprotein leads to cellular surface receptor blockage and imparts immunity to infection by the related leukemia retroviruses. This problem is quite elaborated for chicken endogenous virus RAV-O (7-9).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Isolation of the origin of replication associated with the amplified Chinese hamster dihydrofolate reductase domain.

Autoradiography of restriction digests of DNA labeled in early S phase indicates that replication of the amplified dihydrofolate reductase (DHFR) domain of methotrexate-resistant CHOC 400 cells initiates within a 6.1-kilobase pair (kb) EcoRI-doublet located on the 3' side of the DHFR gene. To localize the DHFR origin fragment, synchronized CHOC 400 cells were either pulse labeled with [3H]thymidine in vivo or permeabilized and incubated with [32P]dATP under conditions that support limited chromosomal DNA replication. The temporal order of replication of amplified fragments was determined by hybridization of the in vivo or in vitro replication products to cloned fragments spanning the earliest-replicating portion of the DHFR domain. At the G1/S boundary, the labeled products derived from the replication of amplified sequences, either in whole or permeabilized cells, are distributed about an amplified 4.3-kb Xba I fragment that maps 14 kb downstream from the DHFR gene. As cells progress through the S phase, bidirectional replication away from this site is observed. These studies indicate that the 4.3-kb Xba I fragment contains the origin of replication associated with the amplified DHFR domain.

Aphidicolin↗
PubMed1990

Evidence that enzymes of a novel aerobic 2-amino-benzoate metabolism in denitrifying Pseudomonas are coded on a small plasmid.

A new pathway for aerobic metabolism of 2-aminobenzoate which proceeds via anthranoyl-CoA has recently been revealed in a Pseudomonas strain KB740. This bacterial strain was found to contain a small 8.1-kbp plasmid pKB740 which appears to harbour the genes encoding for two key enzymes catalyzing the initial reactions of the pathway, 2-aminobenzoate coenzyme A ligase and 2-aminobenzoyl-coenzyme A monooxygenase/reductase. The evidence is as follows: The plasmid content of the culture varied by a factor of ten depending on the growth substrates; it was highest when cells were grown aerobically on 2-aminobenzoate. The plasmid pKB740 could be introduced into Escherichia coli strain JM83 by transformation. Wild-type E. coli and E. coli JM83 are unable to metabolize 2-aminobenzoate whereas the transformed E. coli JM83 cells could grow with this aromatic compound as sole organic substrate and oxidize it completely to CO2. The plasmid recovered from E. coli had the same restriction map as the original plasmid, but was dimerized. The two key enzyme activities were demonstrated in the transformed E. coli in sufficiently high amounts to explain growth. They appear to be regulated on the transcription level by induction; they were formed only during aerobic growth in the presence of 2-aminobenzoate, as in the parent Pseudomonas. The N-terminal amino acid sequence of 2-aminobenzoyl-CoA monooxygenase/reductase was similar to the consensus sequence of the FAD binding site of different flavoenzymes. The data also prove that the enzyme with two flavin functions is a alpha 2 homodimer. Southern blotting of digested chromosomal and plasmid DNA and hybridization against a labelled 15-base oligonucleotide derived from the N-terminal amino acid sequence of 2-aminobenzoyl-CoA monooxygenase/reductase revealed that the gene for this enzyme is coded on the plasmid rather than on the chromosome. The gene was localized on a 3.2-kbp restriction fragment. The formation of 2-aminobenzoyl-CoA monooxygenase/reductase protein in transformed E. coli was demonstrated by Western blotting of proteins of cell extracts separated by SDS/PAGE. The enzyme protein band, which was stained by a procedure based on antibodies against 2-aminobenzoyl-CoA monooxygenase/reductase, was demonstrated in transformed E. coli.

Amino Acid Sequence↗