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M J Getz

Publications and source records attributed to M J Getz.

At least 37 records · Page 2Linked to original sources
PubMed1988

Induction of fibronectin gene transcription and mRNA is a primary response to growth-factor stimulation of AKR-2B cells.

A cDNA library, prepared from poly(A)+ RNA isolated from quiescent AKR-2B cells 4 hr after stimulation with epidermal growth factor in the presence of cycloheximide, was screened to identify RNA transcripts whose abundance is specifically increased as a primary response to growth stimulation. Approximately 40% of the inducible clones detected by this procedure corresponded to either cytoskeletal beta- or gamma-actin genes. One nonactin clone, designated c99, was found to be derived from an 8.5-kilobase RNA whose abundance began to increase as early as 30 min after stimulation. DNA sequencing established the identity of this RNA as fibronectin. Several additional mitogens were then tested and found to efficiently induce fibronectin mRNA. These included fetal calf serum, platelet-derived growth factor, and transforming growth factor type beta. For at least one inducer, fetal calf serum, the increase in mRNA was preceded by an increase in fibronectin gene transcription. This increase was rapid, reaching maximal levels within 10 min, and was accompanied by near-coordinate increases in both c-fos and beta-actin transcription. These results indicate that fibronectin is a member of a class of "early-response" genes, typified by c-fos and including beta-actin, whose rapid expression may be important in mediating cellular responses to peptide growth factors.

Actins↗
PubMed1988

Evidence that the functional beta-actin gene is single copy in most mice and is associated with 5' sequences capable of conferring serum- and cycloheximide-dependent regulation.

Hybridization to synthetic oligonucleotides representing conserved regions in the promoter and first intron of several vertebrate beta-actin genes was used to discriminate between what appears to be a single functional beta-actin gene and numerous pseudogenes in the mouse genome. Sequences derived from the 5' end of this gene were shown to confer serum-inducible expression upon a heterologous reporter gene when transfected into mouse fibroblasts. Moreover, these sequences rendered reporter gene expression superinducible by a combination of serum and cycloheximide. These experiments indicate that the 5' end of the mouse beta-actin gene contains sequence elements which mediate the stimulatory effects of serum growth factors and which are responsive to both positive and negative regulators of gene expression.

Acetyltransferases↗
PubMed1986

Transforming growth factor type beta regulation of actin mRNA.

Stimulation of quiescent cultures of AKR-2B cells with transforming growth factor type beta (TGF beta) resulted in a transitory increase in actin cytoplasmic poly(A) + RNA. Levels of actin mRNA peaked approximately 4-8 hours subsequent to TGF beta addition and approached basal levels by 24 hours. The accumulation of actin transcripts was dose dependent and insensitive to inhibitors of protein synthesis; 1-3 ng/ml TGF beta induced maximal actin gene expression. Actin isotype-specific probes demonstrated that both beta- and gamma-cytoplasmic actins are induced by TGF beta.

Actin Cytoskeleton↗
PubMed1985

Absence of gross change in primary DNA sequence during aging process of mice.

The possibility of age-associated changes in DNA sequence in terms of amplification and rearrangement was examined in mouse spleen, liver and brain using the method of Southern transfer and filter hybridization. The DNA regions studied were at and around nine cloned sequences, most of which are known to move or amplify in certain situations. No detectable age-associated change, however, was observed in all DNA regions studied. These results suggest that widespread DNA sequence rearrangements or amplifications do not occur during the ageing process in mice.

Aging↗
PubMed1984

Specific stimulation of actin gene transcription by epidermal growth factor and cycloheximide.

Stimulation of quiescent AKR-2B mouse embryo cells with epidermal growth factor (EGF) results in a rapid and specific induction of actin mRNA sequences. These mRNAs include those coding for both beta- and gamma-cytoskeletal, but not alpha-skeletal muscle, actin isotypes. Elongation of nascent RNA chains in isolated nuclei (run-off transcription) demonstrates that the mRNA accumulation is preceded by an increase in actin gene transcription. This increase is transient, however, and is followed by a rapid attenuation of transcriptional activity. An inhibitor of protein synthesis, cycloheximide, was also found to induce beta- and gamma-actin mRNA accumulation. Furthermore, the simultaneous addition of EGF and cycloheximide produced a synergistic effect on actin sequences in both steady-state nuclear and polysomal RNA. Run-off transcription experiments demonstrate that this synergistic effect results from an increase in the magnitude and duration of actin gene transcription. It is also specific in that alpha-tubulin gene transcription is not similarly affected. These data suggest the existence of a specific labile repressor of actin gene transcription.

Actins↗
PubMed1983

Discrete regions of sequence homology between cloned rodent VL30 genetic elements and AKV-related MuLV provirus genomes.

Southern blot analyses using reduced stringency hybridization conditions have been employed to search for sequence homologies between rodent VL30 genes and murine leukemia virus (MuLV) proviruses. These constitute two classes of transposon-like elements previously believed to be genetically unrelated. Our results demonstrate that cloned representatives of both ecotropic and xenotropic-like proviruses share discrete regions of sequence homology with VL30 genes of both rat and mouse origin. These regions of homology exist in both 3' and 5' halves of the MuLV genome but do not include extensive portions of the long terminal repeat (LTR) or a 0.4 Kbp segment of the env gene specific for recently acquired ecotropic-type MuLV proviruses. DNA sequencing, however, revealed that the short inverted terminal repeat sequence of MuLV proviral LTRs is almost perfectly conserved at the terminus of an integrated mouse VL30 gene. These results suggest that recombination events with rodent VL30-type sequences occurred during early MuLV evolution. The strong conservation of the inverted terminal repeat sequence may reflect a common integration mechanism for VL30 elements and MuLV proviruses.

Animals↗
PubMed1983

Structure and expression of mouse VL30 genes.

DNA sequencing and blot hybridization analyses have been used to study the structure of a mouse VL30 gene and the molecular nature of VL30-related RNA which is induced upon the stimulation of cultured AKR mouse embryo cells with defined peptide growth factors. An integrated mouse VL30 gene was found to contain identical 601-base-pair long terminal repeats (LTRs) which were themselves terminated in short inverted repeats. The entire VL30 gene was flanked by a 4-base-pair direct repeat of cellular DNA. Thus, VL30 genes are structurally analogous to integrated forms of retrovirus proviruses and certain other classes of mobile genetic elements. The LTR sequence was found to contain putative promoter and polyadenylation signals and generally exhibited little sequence homology to murine leukemia virus proviral LTRs. Certain short regions of sequence conservation, however, were evident, including the inverted terminal repeat, LTR-adjacent regions corresponding to origins of murine leukemia virus proviral DNA synthesis, and a 36-base-pair direct repeat bearing homology to the 72-base-pair direct repeat (enhancer sequence) of the murine leukemia virus-related Moloney sarcoma virus. Upon mitogenic stimulation of quiescent cells with epidermal growth factor and insulin, a major 5.5-kilobase VL30-specific RNA complementary to both LTR and non-LTR sequences was rapidly induced. We conclude that a complete VL30 gene(s) is highly regulated by peptide growth factor binding to specific membrane receptors in these cells.

Animals↗
PubMed1982

Polyadenylylated RNA complementary to a mouse retrovirus-like multigene family is rapidly and specifically induced by epidermal growth factor stimulation of quiescent cells.

Complementary DNA probes prepared from total polysomal poly(A)+RNA populations were used to identify clones of mouse DNA containing sequences whose expression is specifically enhanced after epidermal growth factor (EGF) stimulation of quiescent mouse embryo cells in culture. Three such clones were isolated and used to study changes in the levels of clone-specific poly(A)+RNA in the polysomes of cells after mitogenic stimulation by EGF. RNA complementary to sequences present in these clones increased approximately equal to 10-fold as a fraction of the total poly(A)+RNA by 6 hr after stimulation. All three clones were found by hybridization criteria to contain sequences related to the class of mouse retrovirus or transposon-like elements termed VL30. These VL30-related sequences were further found to be complementary to EGF-inducible poly(A)+RNAs and enhanced expression was detectable as early as 1 hr after EGF stimulation. In contrast, nine additional clones, including an AKR-type murine leukemia provirus DNA clone, contained no detectable VL30 sequence elements and were complementary to poly(A)+RNA species whose relative concentration was essentially constant in quiescent and EGF-stimulated cells. Therefore, VL30 sequence elements appear distinct in that they encompass members whose expression is specifically regulated in response to a defined peptide growth factor.

Animals↗
PubMed1982

Evidence for an early evolutionary origin and locus polymorphism of mouse VL30 DNA sequences.

The VL30 sequences of mouse DNA are a family of sequences with retrovirus-like structure which code for a 30S RNA transcript that can be packaged into the virions of murine leukemia viruses and thereby transmitted from cell to cell. A Southern blot analysis of these sequences revealed that multiple copies are present in the DNA of all mice examined, regardless of species or geographic origin. Considerable locus polymorphism was also apparent, and at least one of these polymorphisms appeared to reflect the differing chromosomal location of a complete VL30 sequence. These data indicated that VL30 elements are not recent additions to the mouse genome and suggested that the evolution of the VL30 multigene family has been accompanied by duplication and dispersion of VL30 sequences to diverse genomic sites. In addition, we reexamined the issue of genetic relatedness between mouse VL30 sequences and a physically similar family of virus-like elements in the rat genome. We found that many, if not all, rat and mouse VL30 loci contain regions of sequence homology. These data suggested that rodent VL30 sequences have evolved from a common ancestral sequence.

Animals↗
PubMed1982

Organization and expression of endogenous virus-like (VL30) DNA sequences in nontransformed and chemically transformed mouse embryo cells in culture.

A cloned mouse DNA fragment containing an endogenous "virus-like" DNA (VL30 DNA) sequence was identified by virtue of its ability to hybridize to the virus-like RNA component of mixed-pseudotype AKR-murine leukemia virus virions, its lack of detectable sequence homology with cloned AKR-murine leukemia virus DNA, and its hybridization to a 5.6 kilobase pair (30S) cellular polyadenylic acid [poly(A)]-containing RNA species. Restriction enzyme mapping of the cloned mouse fragment revealed the presence of a 5- to 6-kilobase pair VL30 DNA segment flanked by non-VL30 segments of approximately 7 and 0.3 kilobase pairs. Southern blot analysis of VL30 DNA sequence organization in the DNA of two nontransformed mouse cell lines (AKR-2B, C3H/10T 1/2) and two chemically transformed derivatives (AKR-MCA, C3H/MCA-58) revealed 15 to 20 bands organized in an apparent strain-specific pattern. Within a given strain, however, no differences were detectable between the nontransformed cells and their chemically transformed counterparts. The expression of VL30 genes in the above cell lines was assayed by hybridization of 32P-labeled poly(A)-containing polysomal RNA to several internal restriction fragments derived from the cloned VL30 DNA sequence. The level of VL30 RNA was enhanced approximately 10-fold in both chemically transformed cell lines as compared to the nontransformed cell lines (under normal growth conditions). In addition, nontransformed AKR-2B cells maintained in the presence of purified epidermal growth factor exhibited similarly enhanced levels of VL30 RNA sequences in polysomal RNA. Since these cells displayed several growth characteristics of transformed cells but, in an epidermal growth factor-dependent and completely reversible fashion, these data suggest that the expression of VL30 genes is not simply incidental to chemically transformed cells but may be related to alterations in growth control.

Animals↗
PubMed1980

Polyadenylate-deficient analogues of poly(A)-containing mRNA sequences in cultured AKR mouse embryo cells.

Five to six percent (by mass) of AKR-2B mouse embryo cell polysomal RNA consists of messenger RNA sequences which may exist in polyadenylated form. In the steady state, however, only 30--40% of these molecules are retained by extensive passage over oligo(dT)-cellulose, the remainder being present in the form of poly(A)-deficient analogues. Within experimental limits, these poly(A)-deficient analogues contain representatives of all poly(A)-containing mRNA sequences in these cells. An analysis of the kinetics of hybridization of cDNA probes enriched for either abundant or rare poly(A)-containing mRNA sequences suggests that the frequency distributions of poly(A)-containing and poly(A)-deficient analogues are dissimilar, and that a relationship exists between the intracellular frequency of a given mRNA sequence and the number of poly(A)-deficient analogues of that sequence. High frequency sequences appear to be enriched in the poly(A)-containing fraction, while low frequency sequences are predominately associated with the poly(A)-deficient fraction, thus, poly(A) may play a role in the regulation of mRNA frequency in the cytoplasm.

Animals↗
PubMed1980

Regulation of endogenous murine leukemia virus-related nuclear and cytoplasmic RNA complexity in C57BL/6J mice of increasing age.

The nucleotide sequence complexity of murine leukemia virus *MuLV)-related RNA has been measured by RNA-complementary DNA hybridization analysis in nuclear and cytoplasmic RNA isolated from liver and brain of low-leukemia-strain C57BL/6J mice of different ages. In these two tissues, an approximate 1.5- to 2-fold increase in the complexity of steadystate nuclear MuLV-related RNA sequences was observed as a function of age. Maximum complexity was observed with nuclear RNA extracts from old mice and corresponded to roughly 70 to 75% of the total MuLV genome. In contrast to the age-related increase in complexity of nuclear MuLV genome was detected in liver and brain steady-state cytoplasmic RNA, irrespective of animal age. These data suggest that control mechanisms regulating the transcription and/or stabilization of nuclear RNA transcripts of endogenous mouse MuLV-related genomes become less stringent with animal age even in low-tumor mouse strains. The data also support the existence of independent posttranscriptional mechanisms which prevent accumulation of these MuLV-related transcripts in steady-state cytoplasmic RNA and which do not seem to be as subject to the relaxation of stringency as a function of age.

Aging↗
PubMed1979

Frequency difference between nuclear and polysomal sequences of poly(A)-containing RNA from cultured AKR mouse embryo cells.

The involvement of post-transcriptional mechanisms in the determination of the frequency distribution of messenger RNA sequences has been studied in cloned mouse embryo cells in culture. Hybridization kinetic experiments between poly(A)-containing RNA and complementary DNA have been used to study the alterations in frequencies which occur in those nuclear poly(A)-containing RNA sequences which are conserved in cytoplasmic poly-ribosomes. Sequences adjacent to nuclear poly(A) tracts are present in a much narrower frequency distribution in the nucleus than in polysomes, with a large proportion of the nuclear sequences present in an average frequency of about one molecule per cell. Few nuclear sequences appear to be present in more than ten copies per cell. A minimum of 70% of these sequences are also found in poly-ribosomal RNA but in greatly altered frequencies. Abundant sequences which comprise a major fraction of the poly(A)-containing polyribosomal RNA are derived from a small fraction of the nuclear poly(A)-adjacent RNA sequences. Very few nuclear poly(A)-adjacent sequences are present in a frequency characteristic of high abundance polysomal sequences. Conversely, poly(A)-containing polyribosomal RNA appears to contain few sequences which are present in as low a frequency as the majority of nuclear poly(A)-adjacent sequences. These observations suggest that post-transcriptional mechanisms play a major role in determining the steady-state frequency of polyribosome-associated messenger RNAs.

Animals↗
PubMed1979

alpha-Amanitin and 5-fluorouridine inhibition of serum-stimulated DNA synthesis in quiescent AKR-2B mouse embryo cells.

AKR-2B mouse embryo cells undergoing the serum-stimulated transition from a quiescent to a proliferating state exhibit an increase in the rate of hnRNA synthesis which appears to be mediated through an increase in the actual number of RNA polymerase II molecules. alpha-Amanitin, administered early in the prereplication interval following stimulation, effectively inhibits hnRNA synthesis, polysomal mRNA accumulation, polyribosome formation, and subsequent DNA synthesis, and cell division. Unexpectedly, alpha-amanitin treatment also produces almost complete inhibition of the synthesis of 45S rRNA precursor and the increase in accumulation of cytoplasmic rRNA following serum stimulation. In order to determine whether the inhibition of new ribosomal synthesis might in itself be sufficient to prevent serum-stimulated DNA synthesis, the effects of 5-fluorouridine (5-FU), a specific inhibitor of 45S rRNA processing, were investigated. If added within eight hours following serum stimulation, 5-FU was found to completely inhibit subsequent DNA synthesis. These results suggest that quiescent AKR-2B cells do not contain a sufficient excess of ribosomes to support the synthesis of proteins which are required for DNA synthesis in response to serum growth factors. Furthermore, an early polymerase II mediated synthesis of mRNA(s) coding for some factor(s) necessary for ribosomal gene transcription may be an essential step in the serum-stimulated synthesis of new ribosomes.

Amanitins↗
PubMed1978

The concept of mRNA abundance classes: a critical reevaluation.

Derivative plots have been constructed for hybridization reactions between polysomal poly(A)-containing RNA and oligo(dT)-primed cDNA. In one method the derivative was calculated directly from the data, and in the other, from a non-linear least squares fit using 9-10 ideal components. In some cases these methods yield very similar results and strongly suggest that the hybridization data support discrete components. Reactions with two and four major components indicate that the often-reported three abundance class model is only one of several possibilities for eukaryotic cells. In other situations neither method strongly suggests the presence of discrete components (in one case even after enrichment of the cDNA population by kinetic fractionation), implying that the components are closely spaced or that the entire mRNA population of those cells may not exist as discrete abundance classes. The universal occurrence of discrete abundance classes should be critically reexamined.

Animals↗