Biomedical subjects

F G Berger

Publications and source records attributed to F G Berger.

At least 37 records · Page 2Linked to original sources

PubMed1994

Evolution of murine alpha 1-proteinase inhibitors: gene amplification and reactive center divergence.

The organization and sequence of genes encoding the alpha 1-proteinase inhibitor (alpha 1PI), a major serine proteinase inhibitor of the mammalian bloodstream, have been compared in several species, including murine rodents (genus Mus). Analysis of gene copy number indicates that amplification of alpha 1PI genes occurred at some time during evolution of the Mus genus, leading to fixation of a family of about three to five genes in several existing species (e.g., M. domesticus and M. saxicola), and only a single gene in others (e.g., M. caroli). A phylogeny for the various mammalian alpha 1PI mRNAs was constructed based upon synonymous substitutions within coding regions. The mRNAs in different murine species diverged from a common ancestor before the formation of the first species lineages of the Mus genus, i.e., about 10-13 million years ago. Thus, alpha 1PI gene amplification must have occurred prior to Mus speciation; gene families were retained in some, but not all, murine species. The reactive center region of the alpha 1PI polypeptide, which determines target protease specificity, has diverged rapidly during evolution of the Mus species, but not during evolution of other mammalian species included in the analysis. It is likely that this accelerated evolution of the reactive center, which has been noted previously for serine proteinase inhibitors, was driven by some sort of a positive Darwinian selection that was exerted in a taxon-specific manner. We suggest that evolution of alpha 1PI genes of murine rodents has been characterized by both modification of gene copy number and rapid reactive center divergence. These processes may have resulted in a broadened repertoire of proteinase inhibitors that was evolutionarily advantageous during Mus speciation.

Amino Acid Sequence↗

PubMed1994

Androgen regulation of gene expression in primary epithelial cells of the mouse kidney.

In proximal tubule cells of the mouse kidney, transcription of a number of genes is induced by androgens. Although a great deal of molecular and genetic information on the induction process has accumulated, the lack of an appropriate cell culture system for DNA transfection studies has hampered efforts to identify and characterize in detail the molecular factors that mediate the response. In the present paper, we have examined a primary renal epithelial cell culture system. We show that in response to androgens, these cells undergo induction of five messenger RNAs that are induced in the intact kidney; thus, the effects of androgens on renal gene expression derive from a direct action of the hormone on proximal tubule cells. The response does not occur in cells from Tfm animals, indicating that androgen receptor is required. Differences in patterns of androgen-inducible messenger RNA expression between mouse species are reflected by similar differences in the cultured cells. Interestingly, the kinetics of induction in culture seem to be distinct from those in the intact kidney, suggesting that a factor or factors differing between the whole animal and tissue culture environments influence the response. Transient transfection experiments with the primary cells showed that the 5'-flanking region of the androgen-regulated RP2 gene, which includes nucleotides -1500 to +42 and contains a glucocorticoid response element that binds the androgen receptor, does not mediate androgen-responsive transcription; thus, this region, and the glucocorticoid response element within it, are either insufficient for, or are not involved in, the gene's response to hormone.

Androgens↗

PubMed1993

Genetic variation in thymidylate synthase confers resistance to 5-fluorodeoxyuridine.

The human colorectal tumor cell line HCT 116 was resident to the cytotoxic effects of 5-fluorodeoxyuridine (FdUrd). The response to FdUrd was increased only slightly by the presence of 10 microM folinic acid (CF). HCT 116 formed FdUMP and CH2H4PteGlu polyglutamates after exposure to FdUrd and CF. The sensitivity to FdUrd correlated well with the extent of TS inhibition. The role of TS in the resistance of the cells to FdUrd was examined. HCT 116 expresses two TS enzymes, which differ in pI. The more basic TS has been detected in only HCT 116 cells. The other TS is identical in pI to the enzymes detected in other human cells. The variant TS differs from the common by His replacement of Tyr at residue 33. The variant TS exhibited a 3-fold lower affinity for FdUMP than the common TS. The enzymes co-expressed in HCT 116 exhibited an FdUMP binding constant similar to that of the variant TS. TS-deficient cells were transfected with cDNAs encoding the two TS polypeptides. Transfectants expressing the variant TS were more resistant to FdUrd cytotoxicity than cells expressing the common TS. Thus, the structural variation in TS reduced enzyme affinity for FdUMP and conferred resistance to FdUrd.

Colorectal Neoplasms↗

PubMed1993

Domains within the mammalian ornithine decarboxylase messenger RNA have evolved independently and episodically.

Ornithine decarboxylase (ODC) is the first enzyme in the polyamine biosynthetic pathway. We have studied the evolutionary history of the mammalian ODC mRNA, focusing on the rate of accumulation of sequence divergence within specific subregions of the molecule. The phylogenetic relationships among the mRNAs from several mammalian species, including two mouse species, rat, hamster, and human, were determined based upon the numbers of synonymous substitutions in pairwise comparisons of mRNA coding regions. The separation times for the mRNAs were very similar to those for the corresponding species, suggesting that ODC is encoded by orthologous genes in the different species. Analysis of divergence patterns in four subregions, or domains, of the mRNA (the 5'-untranslated region, the coding region, and two domains of the 3'-untranslated region) showed that the domains have evolved in a noncoordinate fashion. Furthermore, evolution of each subregion has been episodic, with periods of both rapid and slow sequence divergence. We suggest that the episodic pattern of ODC mRNA evolution may indicate the existence of selection pressures that were exerted in a time- and domain-specific manner during mammalian speciation.

Amino Acid Sequence↗

PubMed1993

Functional effects of a naturally occurring amino acid substitution in human thymidylate synthase.

A major mechanism underlying the cytotoxicity of fluoropyrimidine analogs such as 5-fluorouracil and 5-fluoro-2'-deoxyuridine (FdUrd) occurs via the formation of 5-fluoro-2'-deoxyuridylate (FdUMP), a tight-binding inhibitor of thymidylate synthase (TS). Genetic variation in the structure of the TS molecule is an important determinant of response to fluoropyrimidines, because such variation may affect the binding of FdUMP to the enzyme. Previous studies have shown that the colonic tumor cell line HCT116 expresses two structurally distinct TS polypeptides that differ by the presence of tyrosine or histidine at residue 33. Compared with the Tyr-33 form, the His-33 form confers a 3-4-fold level of FdUrd resistance to cells; this was postulated to be derived from the reduced affinity of the enzyme for FdUMP and N5,N10-methylenetetrahydrofolate, ligands required for the formation of a stable inhibitory complex. In the present study, the Tyr-33 and His-33 forms have been purified to homogeneity, and their properties have been compared in detail. The Km values for dUMP and N5,N10-methylenetetrahydrofolate in the TS reaction were not significantly different between the two enzymes. In contrast, the catalytic efficiency (kcat) was 8-fold lower for the His-33 form. Kinetic and equilibrium binding measurements demonstrated that the dissociation constant for FdUMP binding into the ternary complex was 3-4-fold higher for the His-33 form; this was shown to be due to both a decrease in the rate of FdUMP association with the enzyme and an increase in the rate of FdUMP dissociation from the ternary complex. A TS form containing phenylalanine at residue 33 was created by site-directed mutagenesis and was shown to be very similar to the Tyr-33 enzyme with regard to kcat, pH/activity profile, and effect on FdUrd response. Thus, it is the presence of histidine at residue 33, rather than the absence of tyrosine, that is responsible for the alterations in catalytic and ligand-binding functions exhibited by the His-33 form. Possible mechanisms by which the histidine residue perturbs the structure of the TS active site are discussed.

Base Sequence↗

PubMed1992

Alterations in mRNA translation as a mechanism for the modification of enzyme synthesis during evolution. The ornithine decarboxylase model.

The expression of renal ornithine decarboxylase (ODC) is very different between the two murine species Mus domesticus and Mus pahari. The latter species contains a reduced level of ODC protein in the kidney, yet a normal concentration of ODC mRNA, indicating an alteration in translation, protein degradation, or both. We describe two lines of experimental evidence indicating that the efficiency of ODC mRNA translation is decreased in M. pahari. First, in vivo measurements showed that the rate of biosynthesis of the ODC polypeptide is reduced in M. pahari relative to M. domesticus. Second, polysome analyses indicated that, on average, the ODC mRNA of M. pahari binds fewer ribosomes. These effects, which are kidney-specific, lend support to the notion that renal ODC expression in the two species differs at the level of mRNA translation. A full-length cDNA copy of the ODC mRNA of M. pahari was isolated, and its sequence was compared with that of M. domesticus. In the 5'-untranslated region, which is likely to play an important role in controlling translation, the M. pahari transcript contains several single-base changes, as well as a 12-base deletion. These changes confer a distinct predicted secondary structure to this region of the mRNA and may be involved in reduced translation. We conclude that during evolution, modifications that alter the biosynthesis of specific proteins can occur at the translational level; these modifications exert their effects on translation in a transcript- and tissue-specific manner.

Amino Acid Sequence↗

PubMed1992

The evolution of coexisting highly divergent LINE-1 subfamilies within the rodent genus Peromyscus.

Two distinct members of the LINE-1 (L1) family in Peromyscus were characterized. The two clones, denoted L1Pm55 and L1Pm62, were 1.5 kb and 1.8 kb in length, respectively, and align to the identical region of the L1 sequence of Mus domesticus. Sequence similarity was on the order of 70% between L1Pm55 and L1Pm62, which approximates that between either Peromyscus sequence and Mus L1. L1Pm62 represents a more prevalent subfamily than L1Pm55. L1Pm62 exists in about 500 copies per haploid genome, while L1Pm55 exists in about 100 copies. The existence of major and minor subpopulations of L1 within Peromyscus is in contrast to murine rodents and higher primates, where L1 copy number is on the order of 20,000 to 100,000, and where levels of intraspecific divergence among L1 elements are typically less than 15-20%. Additional Peromyscus clones are similarly divergent from both L1Pm62 and L1Pm55, implying the existence of more than two distinct L1 subfamilies. The highly divergent L1 subfamilies in Peromyscus apparently have been evolving independently for more than 25 million years, preceding the divergence of cricetine and murine rodents. Investigations of the evolution of L1 within Peromyscus by restriction and Southern analysis was performed using species groups represented by the partially interfertile species pairs P. maniculatus-P. polionotus, P. leucopus-P. gossypinus, and P. truei-P. difficilis of the nominate subgenus and P. californicus of the Haplomylomys subgenus. Changes in L1 and species group taxonomic boundaries frequently coincided. The implications for phylogeny are discussed.

Animals↗

PubMed1992

A naturally occurring tyrosine to histidine replacement at residue 33 of human thymidylate synthase confers resistance to 5-fluoro-2'-deoxyuridine in mammalian and bacterial cells.

Structural changes in the macromolecular targets of pharmacological agents can result in alterations in the efficacy of these agents. In previous studies, we identified a variant structural form of thymidylate synthase (TS) that is associated with relative resistance to 5-fluoro-2'-deoxyuridine, in a human colonic tumor cell line. We now report on the use of DNA transfer techniques to examine directly the effects of each TS form on drug response. TS cDNA constructs, corresponding to the normal or variant TS mRNA, were expressed in Chinese hamster lung cells or in Escherichia coli, and response to 5-fluoro-2'-deoxyuridine was determined. We observed that expression of the variant TS, which differs from the normal form by a tyrosine to histidine substitution at residue 33, confers a 4-fold level of drug resistance in the mammalian cells, as well as in bacteria. The possible role of Tyr-33 in 5-fluoropyrimidine-mediated inhibition of TS is discussed.

Amino Acid Sequence↗

PubMed1991

Acute phase mediated change in glycosylation of rat alpha 1-acid glycoprotein in transgenic mice.

Transgenic mouse lines carrying the gene for rat alpha 1-acid glycoprotein (AGP) express the protein in the plasma at concentrations equal to or exceeding that of acute phase rats. Owing to the high basal level, these transgenic mice represent a unique experimental system for defining the largely unknown function of AGP. Since the carbohydrate moiety of AGP has been found to be changed during acute phase and the oligosaccharide structure to be important for immunomodulating activity of the protein, the rat AGP in transgenic mice was characterized by lectin-affinity immuno-electrophoresis. Unlike in the rat, the AGP in the transgenic mouse plasma consisted primarily of strongly concanavalin A-reactive forms. Acute phase mediated a several-fold increase in the total plasma concentration of AGP concomitant with a shift toward moderately concanavalin A-reactive forms. A similar change in concanavalin A-reactive forms was observed for the endogenous acute phase plasma protein haptoglobin. To define the role of inflammatory factors in AGP production, primary cultures of hepatocytes were prepared. In contrast to in vivo, the AGP recovered from tissue culture medium represented primarily the concanavalin A-non-reactive form. Treatment of the cells with recombinant human interleukin-1, interleukin-6 and dexamethasone stimulated the production of concanavalin A-reactive AGP forms. The data indicate that the glycosylation pattern of plasma-resident AGP is modulated by acute phase, but that the profile of AGP forms does not coincide with that secreted by hepatocytes in tissue culture. This finding demands an assessment of which of the possible glycosylated forms of AGP is functionally significant in vivo.

Animals↗

PubMed1991

Evolution of messenger RNA structure and regulation in the genus Mus: the androgen-inducible RP2 mRNAs.

The RP2 gene is one of several genes that are regulated by androgens in the mouse kidney. Previous studies have demonstrated that androgen inducibility of RP2 transcription varies among species within the genus Mus, indicating extensive evolutionary modification of the participating regulatory elements. Thus, while a five-fold induction of transcription occurs in M. domesticus, none is detectable in M. hortulanus or M. caroli. In the present paper, we have sequenced cDNAs representing the RP2 mRNAs of M. caroli and M. saxicola and have compared them with each other and with M. domesticus. Several findings from the sequence comparisons indicate that the encoded 41-kD polypeptide is physiologically functional. First, divergence within noncoding regions of the mRNAs exceeds that within coding regions. Second, the 357-codon open reading frame has been maintained among the species, with approximately 90% of the amino acid replacements being conservative. Finally, substitution rates at synonymous sites within the coding regions are from twofold to threefold greater than those at nonsynonymous sites. The genetic elements responsible for variations in RP2 inducibility among species were studied by cis/trans analyses of mice heterozygous for RP2 alleles, using a primer extension assay to measure expression of species-specific mRNAs. The results show that the absence of transcriptional induction in M. hortulanus is due to a cis-acting genetic element, while that in M. caroli is due to a trans-acting element. Thus, the androgen-resistant RP2 phenotypes of these two species derive from distinct genetic events.

Animals↗

PubMed1991

Androgen modulation of DNA-binding factors in the mouse kidney.

Transcription of the RP2 gene in the mouse kidney is induced by androgens. This induction is species specific within the genus Mus. For example, the gene responds to androgens in Mus domesticus, but is refractory to hormone in the distantly related species M. caroli. In the present report we have characterized DNA-binding factors that recognize the 5' flanking region of the RP2 gene. One factor (termed RPBF-1) binds a DNA fragment spanning the region between -157 and -311 relative to the transcriptional start site. RPBF-1 is present in kidney nuclear extracts from both control and androgen-treated M. domesticus as well as from control M. caroli; however, in the latter species a distinct factor (termed RPBF-2) is induced by androgens and replaces RPBF-1. The androgen-dependent replacement of RPBF-1 by RPBF-2 is specific to the kidney of M. caroli. DNase-1 footprinting analyses indicate that the two factors recognize distinct, yet overlapping, regions of the RP2 promoter: RPBF-1 binds the region between -247 and -269, while RPBF-2 binds the region between -265 and -290. The RPBF-2-binding site contains a sequence that is homologous to that recognized by nuclear factor-1 (NF-1), suggesting that RPBF-2 is a NF-1-like factor. This is supported by competition experiments with synthetic oligonucleotides corresponding to the NF-1-binding site within the adenovirus origin of replication. Thus, androgens can modulate, in a species- and tissue-specific manner, DNA-binding factors that recognize promoter regions of genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Inducible and tissue-specific expression of rat alpha-1-acid glycoprotein in transgenic mice.

alpha-1-Acid glycoprotein (AGP), which is produced in the mammalian liver and secreted into the blood-stream, is regulated by steroid hormones and by mediators of the acute phase response. In vitro transfection studies have shown that the response to glucocorticoids requires a cis-acting regulatory element, termed the glucocorticoid response element, that is located within 120 bp of the transcriptional start-site of the gene; induction by the acute phase mediators requires a different element, termed the distal regulatory element (DRE), that is located about 5 kb upstream of the start-site. To determine if these elements function in vivo, we have produced and characterized transgenic mice containing rat AGP gene constructs with and without the DRE. Five transgenic lines were produced from a 9.5-kb genomic AGP containing 4.7 kb of the 5' flanking region; this construct lacks the DRE. Another transgenic line was derived from a 10.7-kb clone that contains 5.3 kb of 5' flanking sequences including the DRE. All transgenic mice produced high levels of immunologically detectable rat AGP in the circulation, comparable to or in excess of that found in normal rats. There were correspondingly high concentrations of rat AGP transcripts in the liver. Transgene expression in all lines was induced in response to dexamethasone and during acute inflammation resulting from LPS treatment. The DRE-containing transgene underwent a greater induction in response to LPS than to dexamethasone; the transgene lacking the DRE responded similarly to both treatments. In cultured primary hepatocytes, the DRE-containing transgene was induced by the acute phase cytokines IL-1 and IL-6, and by dexamethasone, administered individually or in combination; the transgene lacking the DRE responded only to dexamethasone, and was not affected by the peptide hormones. Together, these results provide in vivo evidence supporting the notion that a minimum of two upstream sequences are responsible for the inflammatory induction of rat AGP. One element, which is located within the smaller 9.7-kb restriction fragment, is responsive to glucocorticoids and is likely to be the glucocorticoid response element located close to the transcriptional start site. The other element, the DRE, is located much further upstream and is responsible for imparting responsiveness to the acute phase cytokines.

Animals↗

PubMed1990

Highly conserved upstream regions of the alpha 1-antitrypsin gene in two mouse species govern liver-specific expression by different mechanisms.

alpha 1-Antitrypsin (AT), the major elastase inhibitor in mammalian serum, is produced primarily in the liver. We have characterized AT gene structure and expression in the mouse species Mus caroli, which expresses high levels of AT in the kidneys as well as in the liver. Analysis of cDNA and genomic clones showed that the AT gene in M. caroli exhibits high sequence homology (greater than 90%) to the gene in laboratory mice (M. domesticus) throughout the coding and 5'-flanking regions. Despite this extensive sequence conservation, the functional organization of cis-acting regulatory elements governing liver-specific expression is strikingly different between these species. Transient-transfection assays showed that the proximal region of the M. caroli promoter (i.e., between -120 and -2 relative to the transcriptional start site) is 10-fold more active than the analogous region of M. domesticus in driving the expression of an indicator gene in cultured liver cells. The increased activity of the proximal region of the M. caroli AT promoter appears to be the result of one or both of the two base substitutions at positions -46 and -48. The weak proximal promoter in M. domesticus is compensated for by the presence of upstream, liver-specific enhancers between -199 and -520; the analogous region in M. caroli is inactive. Thus, during the course of evolution, the modest 7% sequence divergence that has occurred between the 5'-flanking regions of the AT genes in these two species has generated distinct, yet equally effective, modes of hepatocyte-specific expression.

Amino Acid Sequence↗

PubMed1990

Single amino acid substitution defines a naturally occurring genetic variant of human thymidylate synthase.

Previously, we identified an altered structural form of thymidylate synthase (TS) in a human colonic tumor cell line. This form, which is encoded by a variant structural gene, renders cells relatively resistant to 5-fluoro-2'-deoxyuridine as a result of the reduced affinity of the enzyme for the active metabolite 5-fluoro-2'-deoxyuridylic acid. We have isolated a cDNA clone specific to the altered TS and have determined its sequence. Two point mutations distinguish the normal from the altered TS mRNAs. One, a (A----G) change, is located within the 3'-untranslated region; the other, a T----C change within the amino acid-coding region, predicts replacement of tyrosine by histidine at residue 33 of the polypeptide. This sequence change was confirmed by direct analysis of cDNA amplified by the polymerase chain reaction and was further verified using allele-specific oligonucleotides as probes in Northern blots. These results, along with studies by other laboratories showing Tyr33 to be evolutionarily conserved, suggest that this residue plays an important role in TS function.

Amino Acid Sequence↗

PubMed1989

Assignment of a gene encoding ornithine decarboxylase to the proximal region of chromosome 12 in the mouse.

Ornithine decarboxylase (ODC), the first enzyme in the polyamine biosynthetic pathway, is encoded by at least one member of a multi-gene family in the mouse. Analysis of a polymorphism in ODC structure in recombinant inbred strains has enabled assigning a functional ODC structural gene (Odc) to the proximal region of mouse chromosome 12 between Apob and Es25. Linkage of Odc to Apob and Ah is conserved in the mouse and human genomes.

Animals↗

PubMed1989

A family of type I keratin genes and the homeobox-2 gene complex are closely linked to the rex locus on mouse chromosome 11.

Type I and type II keratins are major constituents of intermediate filaments that play a fundamental role in the cytoskeletal network. By using both somatic cell hybrids and conventional and interspecific linkage crosses, several genes encoding type I keratins, including the epidermal keratin K10, were shown to be closely linked to the homeobox-2 complex and the rex locus on mouse chromosome 11. The absence of crossovers between type I keratin-encoding genes and rex (N = 239), a locus affecting hair development, raises the possibility that mutations at rex and neighboring loci affecting skin and hair development involve type I keratin genes.

Alleles↗

PubMed1989

Molecular genetics of androgen-inducible RP2 gene transcription in the mouse kidney.

Androgen control of the RP2 gene in the mouse kidney has been modified during evolution. In inbred mice (Mus domesticus), the concentrations of mRNAs encoded by RP2 undergo a 10- to 12-fold induction in response to testosterone; in other Mus species (e.g., Mus hortulanus and Mus caroli), induction ranges from none to about two- to fourfold. In this communication, we show that androgens induced RP2 transcription in M. domesticus, although this induction may not have fully accounted for the increase in mRNA levels. Reduced mRNA inducibility in M. hortulanus and in several other species was associated with an absence of transcriptional induction. Analysis of an interspecies backcross population indicated that the difference in RP2 inducibility between M. domesticus and M. hortulanus was due to a single Mendelian locus tightly linked (0 of 47 recombinants) to RP2. The RP2 gene was found to contain at least two promoters, only one of which was highly sensitive to testosterone. These results indicate that induction of the RP2 mRNAs, as well as interspecies variations in RP2 inducibility, are primarily a consequence of effects on this promoter.

Animals↗

PubMed1989

Androgen-regulated gene expression.

While a great deal of knowledge on the mechanisms of steroid hormone regulated gene expression now exists, specific information relating to androgens is lacking. A number of experimental systems have been developed and show promise as models for molecular studies of androgen regulation. Further development of these models must address the issue of whether or not the androgen receptor behaves similarly to other steroid receptors. This will require progress in the purification of the receptor itself or the cloning of its gene. Several features of androgen-regulated gene expression in the mouse kidney are applicable to a number of important biological problems. First, the presence of a variety of inducible mRNAs, whose responses to androgen are controlled at several genetic and molecular levels, should be valuable in obtaining basic information on mechanisms of gene regulation by hormones. Second, a genetic approach, for which the mouse is a convenient organism, will enable identification of novel regulatory elements that are responsible for variations in the hormonal activation of genes and that drive the evolution of species-specific gene expression phenotypes. Finally, the trophic effects of androgens in the mouse kidney afford opportunities to study a gene expression phenotype that is involved in cell growth; it will be of interest to determine how androgen induction of gene expression in kidney proximal tubule cells relates to the hormone-mediated growth of these cells.

Alcohol Dehydrogenase↗