Biomedical subjects

G R Stark

Publications and source records attributed to G R Stark.

At least 127 records · Page 7Linked to original sources

PubMed1988

Excess antisense RNA from infectious recombinant SV40 fails to inhibit expression of a transfected, interferon-inducible gene.

SV40-based infectious virus constructs were used to produce a high copy number of full-length antisense RNA in essentially every cell in a population. Chloramphenicol acetyltransferase (CAT) cDNA was placed in either the sense or antisense orientation relative to the SV40 early promoter in helper-free recombinant virus. RNA synthesized at high levels from the antisense virus was without effect on the expression of a stably-transfected CAT mini-gene controlled by an interferon-inducible promoter in monkey CV1 and large T antigen-expressing tsCOS cells. In double infection experiments the antisense RNA was similarly without effect on expression from CAT cDNA placed in the sense orientation in a second virus vector. No activation of the ppp(A2'p)nA(n greater than or equal to 2) system was observed after interferon treatment in either type of experiment. There was no evidence, therefore, for the formation of double-stranded (ds)RNA. It can be concluded that a large excess of a full-length antisense RNA is not necessarily sufficient to cause inhibition of gene expression even when interferon treatment is used to enhance any effect of dsRNA.

Acetyltransferases↗

PubMed1988

Interferon response element of the human gene 6-16.

1046 base-pairs (bp) of genomic DNA spanning the first exon of the human alpha/beta-interferon (IFN)-inducible gene 6-16 have been analysed for their role in induction. The whole gene or 5'-flanking deletion derivatives of it were assayed for inducibility in populations of stably transfected mouse cells. 5'-Flanking DNA fragments were assayed for their ability to confer inducibility on a reporter gene in stably and transiently transfected mouse and human cells. The data suggest that a 39 bp sequence is sufficient to confer transcriptional inducibility and can account in large part for the response of 6-16. Two copies of this sequence, one of which contains a dinucleotide insert, are located in tandem 88 bp upstream of the 6-16 transcriptional initiation site. For at least one of the repeat units the 5' limit of a subregion required for induction lies in the sequence GGGAAAAT. The motif GGAAA occurs in several well characterized enhancers. Furthermore, one residue 3' of the GGAAA there is a second motif, TGAAACT, which is conserved in the regulatory regions of other IFN-induced genes. In gel retardation assays the oligonucleotide GGGAAAATGAAACT competes with the repeat element for binding to IFN-modulated protein(s) but a mutated oligonucleotide, GGGAAAATGACACT does not. These results identify an alpha/beta IFN response element partially homologous to those described previously for the genes of the MHC complexes.

Animals↗

PubMed1988

A quantitative method for analyzing specific DNA sequences directly from whole cells.

A quick, accurate assay for specific DNA sequences is described in which whole cells are treated with 0.4 M sodium hydroxide at 80 degrees C. DNA is relatively resistant to alkaline hydrolysis, whereas proteins and RNA are degraded rapidly. The DNA in NaOH is then transferred through a slot directly onto a nylon membrane and hybridized with a probe. Since the procedure is so simple, many samples can be analyzed in a short time. A single-copy gene can be detected in as few as 1000 cells and, since the DNA from 10(5) cells can be loaded through a single slot, the sensitivity is sufficient to detect one specific DNA sequence per 100 cells. Accurate quantitative analysis can be achieved by normalizing the amount of DNA available for hybridization in each slot, using a probe derived from total DNA.

Animals↗

PubMed1988

Preferential amplification of rearranged sequences near amplified adenylate deaminase genes.

In a previous study of three independent families of mutants selected for overproduction of adenylate deaminase (AMPD), we were not able to isolate a cDNA probe for the gene and so could not demonstrate its amplification directly. In addition to overproduction of AMPD, four proteins of unknown function, designated W, X, Y1, and Y2, accumulated, and by using the corresponding cDNA probes, we demonstrated amplification of all four genes. In independent mutant clones, sometimes all and sometimes only a subset of these genes were amplified. Assuming that all five genes are linked, the pattern of their coamplification suggested a genetic map in which AMPD lies between W and Y1. We show here that a two-step chromosome walk joins the W and Y1 genes, that the AMPD gene is the only expressed sequence between them, and that its amplification is indeed responsible for overproduction of the AMPD protein. In the course of this work, we cloned and studied two novel joints which mark rearrangements on either side of the AMPD gene. Each joint was generated independently in a single first-step mutant at single or low copy number. Remarkably, each joint was amplified preferentially in every second- and third-step mutant derived from the first-step line in which it was originally present, suggesting that the two independent rearrangements each generated amplification-prone structures.

AMP Deaminase↗

PubMed1987

Hamster cells with increased rates of DNA amplification, a new phenotype.

Baby hamster kidney (BHK) cells selected simultaneously with N-phosphonacetyl-L-aspartate (PALA) and methotrexate (MTX) gave rise to doubly resistant colonies at frequencies 20 to 260 times greater than the product of the independent frequencies found with PALA or MTX alone. Double resistance was due to amplification of both target genes, CAD and DHFR. Four independent doubly resistant "MP" lines were selected and characterized. Cells resistant to coformycin, pyrazofurin, or ouabain were generated from all four MP lines at rates up to 25 times greater than the rates for BHK cells. These three drugs select cells that have amplified the genes for their target enzymes. Therefore, we conclude that the four MP lines have an amplificator phenotype. All four grew much more slowly than BHK cells, indicating that the amplificator phenotype may be linked to significant defects in metabolism or cell division.

Amides↗

PubMed1986

Regulation of carbamoyl phosphate synthetase-aspartate transcarbamoylase-dihydroorotase gene expression in growing and arrested cells.

We have studied expression of the carbamoyl-P synthetase-aspartate transcarbamoylase-dihydroorotase (CAD) gene in growing or confluent Syrian hamster cells, and also in cells arrested by depriving them of serum or restimulated by adding fresh serum. In contrast to other biosynthetic enzymes such as dihydrofolate reductase, the amount of the CAD enzyme decreased very little when growth was arrested, as judged by the small change in aspartate transcarbamoylase activity. However, the level of CAD mRNA was about 10-fold lower in arrested cells than in growing cells. The rate of transcription of CAD, measured by the nuclear run-off technique, decreased roughly in parallel with the decrease in the steady-state level of the mRNA, suggesting that control of transcription is probably an important element in the overall regulation of CAD gene expression. Expression of a different gene which lies near the 5' end of CAD changed in parallel with that of CAD, but expression of two other genes in the same region was insensitive to the growth state of the cells. Therefore, control of gene expression as a function of growth was specific rather than a reflection of more general changes in chromatin structure in the vicinity of CAD.

Amidohydrolases↗

PubMed1986

Characterization of a human gene inducible by alpha- and beta-interferons and its expression in mouse cells.

An intact interferon-inducible gene has been isolated from a cosmid library of human genomic DNA. The gene (designated 6-16) encodes a mRNA of approximately 1 kb which is induced well by alpha- and beta- but poorly by gamma-interferons. Genomic and cDNA sequences indicate that the gene contains five exons, and that the mRNA encodes a hydrophobic polypeptide of 130 amino acids with a putative NH2-terminal signal sequence. The 5' end has been identified by primer extension. The corresponding genomic DNA contains a TATA box 20 nucleotides upstream of the putative transcription initiation site. After transfection of the human genomic cosmid into mouse Ltk- cells, human 6-16 mRNA is expressed in response to mouse alpha- and beta- but not gamma-interferons with the same kinetics and dose-response as in the human cells. No such expression is observed in response to human interferons. It can be concluded that the human cosmid DNA contains all of the sequences necessary for alpha- and beta-interferon-induced gene expression and that the mechanisms governing such expression are conserved between murine and human cells.

Amino Acid Sequence↗

PubMed1986

Structure of DNA formed in the first step of CAD gene amplification.

Thirty-three independent mutant cell lines were selected in single steps for resistance to low concentrations of N-(phosphonacetyl)-L-aspartate and the structure of their amplified DNA was probed, using a set of recombinant phage and cosmids containing a total of 380 kb of amplified DNA. In all 33 cell lines, the selected CAD gene and at least 65 kb of flanking DNA were amplified, an average of 2.6-fold. Six other regions of DNA were co-amplified in all 33 mutants, but sometimes to a different extent than CAD. Novel joints, marking recombinations which link amplified regions to each other, were found surprisingly rarely. There were only three within the 380 kb of DNA sequence examined in the total of 33 cell lines. Each novel joint was present in only one copy per cell, was found in a different cell line and was homologous to a different probe. The low frequency of novel joints is consistent either with very large amplified regions in the single-step mutants, possibly 10,000 kb of co-amplified DNA for each copy of the CAD gene, or with a strong bias against recombination in the cloned sequences used as probes. Our previous finding that CAD probes hybridize in situ to unusually large chromosome arms in several single-step mutants is most consistent with the first possibility.

Animals↗

PubMed1986

Charomids: cosmid vectors for efficient cloning and mapping of large or small restriction fragments.

Charomids are cosmid vectors up to 52 kilobases (kb) long, bearing 1-23 copies of a 2-kb spacer fragment linked in head-to-tail tandem arrays. Like cosmids and lambda phage, charomids can be packaged in vitro for efficient introduction into bacteria. Charomids contain a polylinker with nine unique restriction sites for cloning and can be used without preparing vector arms. Using a charomid of appropriate size, one can clone inserts of any size up to 45 kb. For example, charomid 9-36 (9 cloning sites, 36 kb long) is too small to be packaged efficiently without an insert and can be used to clone fragments of 2-16 kb. The structure of charomids facilitates restriction mapping of the insert DNA and, after cloning, all the spacer fragments can be removed easily. After enrichment by size fractionation in an agarose gel, a specific single-copy genomic sequence can be cloned rapidly from approximately 3 micrograms of DNA. Using charomid 9-36, we have cloned and mapped an amplified novel DNA fragment from a cell line resistant to N-(phosphonoacetyl)-L-aspartate and carrying about 100 copies of the CAD (carbamoyl-phosphate synthetase/aspartate carbamoyltransferase/dihydroorotase) gene. The fragment lies at the center of an inverted duplication of this gene.

Base Sequence↗

PubMed1986

Interferon-induced gene expression in wild-type and interferon-resistant human lymphoblastoid (Daudi) cells.

Interferon-induced gene expression was analyzed in wild-type and interferon-resistant Daudi cells. Two classes of alpha-interferon-induced mRNAs and proteins were observed: those that were similarly induced in both types of cell and those that were induced only in the wild-type cells. Furthermore, the level of c-myc mRNA decreased in the wild-type but not in the resistant cells. This differential control in the wild-type and resistant cells indicates that there must be either functionally distinct alpha-interferon receptors or more than one pathway leading to altered gene expression triggered by a single receptor.

Cell Division↗

PubMed1986

DNA amplification in drug resistant cells and in tumours.

DNA amplification is a pervasive phenomenon in continuous cell lines and in tumours. It can occur in many different parts of the genome with high frequency, and a very large amount of DNA, probably including many genes, is often involved in a single amplification event. In tumours, it is likely that a common mechanism underlies the amplification of genes which confer drug resistance and genes which give a growth advantage to the tumour. DNA amplification may be caused by defects in the regulation of DNA replication, and similar defects may lead to other chromosomal abnormalities such as translocations, inversions and heteroploidy. Two recent observations should facilitate identification and study of the underlying defects: agents or treatments which damage DNA can increase the rate of amplification, and mutant cell lines can be selected which amplify their DNA at a greatly increased frequency.

Animals↗

PubMed1985

Differential regulation of interferon-induced mRNAs and c-myc mRNA by alpha- and gamma-interferons.

The extent and kinetics of induction have been determined for eight mRNAs induced by alpha- and gamma-interferons in HeLa cells. The mRNAs which code for a 2-5A synthetase, metallothionein II, HLA class I antigen and five unknown proteins were induced 2 - greater than 100-fold by alpha-interferons. In the continued presence of alpha-interferon some mRNAs were maintained at the induced levels until at least 40 h, whereas others were induced only transiently. When the effects of alpha- and gamma-interferons were compared, the induced levels and kinetics were very similar for one mRNA (1-8) but were significantly different for the others. One mRNA (6-16) was induced more than 100-fold by alpha-interferon but not significantly by gamma-interferon. Parallel analysis of the level of c-myc mRNA showed it to decrease twofold in response to alpha-interferon, but to increase more than threefold in response to gamma-interferon, despite a more profound inhibition of cell growth by the latter. There must, therefore, be differences in how the levels of different mRNAs are sustained by alpha-interferons and how alpha- and gamma-interferons regulate the levels of the same mRNAs.

Cloning, Molecular↗

PubMed1985

Abundant nuclear ribonucleoprotein form of CAD RNA.

Transcripts of the CAD gene in Syrian hamster cells are as abundant in the nucleus as in the cytoplasm. This was shown by in situ hybridization of whole cells and by solution and blot hybridization of subcellular fractions. Similar results were obtained both for wild-type cells and for a mutant containing amplified CAD genes in which the level of CAD RNA is 150-fold greater. CAD nuclear RNA is indistinguishable from mature mRNA by gel electrophoresis and blot hybridization. Discrete higher-molecular-weight precursors are undetectable, although the persistence of a short length of intervening sequence in the otherwise fully processed RNA is not excluded. CAD RNA is released from nuclei by sonication in physiological conditions in a ribonucleoprotein form that sediments as a broad peak at about 200S in a sucrose gradient. CAD sequences extracted from nuclei by treatment with EDTA and RNase are found in the 30S particles previously described.

Animals↗

PubMed1985

Construction of a cDNA to the hamster CAD gene and its application toward defining the domain for aspartate transcarbamylase.

cDNA complementary to hamster mRNA encoding the CAD protein, a multifunctional protein which carries the first three enzymes of pyrimidine biosynthesis, was constructed. The longest of these recombinants (pCAD142) covers 82% of the 7.9-kilobase mRNA. Portions of the cDNA were excised and replaced by a lac promoter-operator-initiation codon segment. The resultant plasmids were transfected into an Escherichia coli mutant defective in aspartate transcarbamylase, the second enzyme of the pathway. Complementation of the bacterial defect was observed with as little as 2.2 kilobases of cDNA sequence, corresponding to the 3' region of the mRNA. DNA sequencing in this region of the hamster cDNA reveals stretches which are highly homologous to the E. coli gene for the catalytic subunit of aspartate transcarbamylase; other stretches show no homology. The highly conserved regions probably reflect areas of protein structure critical to catalysis, while the nonconserved regions may reflect differences between the quaternary structures of E. coli and mammalian aspartate transcarbamylases, one such difference being that the bacterial enzyme in its native form is allosterically regulated and the mammalian enzyme is not.

Amidohydrolases↗

PubMed1984

Induction of human mRNAs by interferon.

We have cloned a set of seven cDNAs corresponding to mRNAs induced by type I interferon in human cells, and are using these probes to characterize the response to interferon in detail. We have studied the kinetics of induction of each mRNA in three different human cell lines, the sensitivity of induction to cycloheximide, the effect of withdrawing interferon and the response of a receptor-positive interferon-resistant cell line.

Cell Line↗

PubMed1984

Simian virus 40-infected, interferon-treated cells contain 2',5'-oligoadenylates which do not activate cleavage of RNA.

2',5'-oligoadenylates can be assayed sensitively in cell extracts by use of an antiserum having maximum specificity for any compound containing the moiety -pA2'pA2'pA-. These compounds reached high concentrations (25-2000 nM) in monkey CV-1 cells after infection with simian virus 40 (SV40) and treatment with human leukocyte interferon. The levels were highest late in infection and increased in parallel with the accumulation of SV40 late messenger RNAs. Alone, neither interferon nor SV40 caused the 2',5'-oligoadenylate concentrations to increase above the levels present in untreated CV-1 cells, 3 nM or less. Analyses by high performance liquid chromatography revealed little or no (p)pp(A2'p)2A or (p)pp(A2'p)3A, and the extracts showed only very low activity in functional assays with ppp(A2'p)nA-dependent nucleases, equivalent to 3 nM ppp(A2'p)3A or less. Some of the 2',5'-oligoadenylates eluted in the positions of the nonphosphorylated "cores," (A2'p)nA, and a substantial fraction was found in several peaks intermediate between ppp(A2'p)3A and cores. The positions of most of these peaks did not change when digestion with alkaline phosphatase was performed before chromatography, indicating that most of the 2',5'-oligoadenylates lack exposed phosphate groups. In contrast to the effects of infection with SV40, addition of poly(I) X poly(C) to interferon-treated CV-1 cells led to accumulation of high levels (up to 3000 nM) of 2',5'-oligoadenylate-5'-di- or triphosphates capable of activating the ppp(A2'p)nA-dependent ribonuclease.

Adenine Nucleotides↗

PubMed1984

Preparation, characterization, and use of an antiserum specific for 2',5'-oligoadenylates.

Periodate-oxidized ppp(A2'p)3A was coupled in high yield to an acyl hydrazide derivative of bovine serum albumin and the conjugate was used to immunize rabbits. Several potent and specific antisera were obtained, and one was tested extensively. Fifty per cent of the bound probe ppp(A2'p)3A3'[32P]pCp was displaced by compounds containing the moiety -pA2'pA-at concentrations of 20-60 nM and by compounds containing the moiety -p(A2'p)2A- at 1 nM. The 3',5'-oligoadenylate (A3'p)3A was bound more than 10,000 times less tightly than (A2'p)3A. The antiserum can be used in competition assays in which the amount of an antibody-probe complex is measured after binding to Millipore filters or to polystyrene beads. These assays allow the clear detection of as little as 20 fmol (20 microliters of 1 nM) of 2',5'-oligoadenylates.

Adenine Nucleotides↗

PubMed1984

An improved method for purifying 2',5'-oligoadenylate synthetases.

We describe a new, rapid, and convenient procedure for purifying 2',5'-oligoadenylate synthetases, employing precipitation with ammonium sulfate, fractionation by gel filtration, rapid binding to poly(I) X poly(C) cellulose, and elution with 0.35 M KCl. Unlike previously published methods, the procedure does not require sedimentation of the enzyme at 200,000 X g. Therefore, it is more general and more likely to succeed with synthetases extracted from a variety of cells or tissues, or from different subcellular fractions. We have purified the enzymes from two sources to apparent homogeneity, about 2500-fold from the cytoplasm of HeLa cells in 40% yield and more than 400,000-fold from the cytoplasm of rabbit reticulocytes in 25% yield. The specific activity of the HeLa enzyme is about 4 times higher than reported previously. The physical and functional properties of the pure enzymes are very similar to those reported by others for preparations of 2',5'-oligoadenylate synthetase from rabbit reticulocytes, mouse L cells, and human HeLa cells. A new affinity matrix was prepared by linking periodate-oxidized poly(I) X poly(C) to a hydrazide derivative of finely divided cellulose. Poly(I) X poly(C) cellulose binds about twice as much synthetase as the corresponding amount of poly(I) X poly(C) paper and activates the bound enzyme about three times better.

2',5'-Oligoadenylate Synthetase↗