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M Altmann

Publications and source records attributed to M Altmann.

At least 55 records · Page 3Linked to original sources
PubMed1993

Regulation of translation initiation and modulation of cellular physiology.

Translational control of gene expression is an important regulatory mechanism in cellular physiology. In eukaryotes, ribosomes can initiate translation by two different mechanisms: a majority of mRNAs undergo cap-dependent initiation at their extreme 5'-ends, but initiation can occur internally in some mRNAs. A number of important cellular responses, such as entry into a proliferative state and adaptation to changing nutrient levels, are mediated by changes in the mechanism of translation initiation of specific mRNAs. This article discusses new insights into control of gene expression gained through studies of regulation of eukaryotic translational initiation.

Cell Division↗
PubMed1993

TIF4631 and TIF4632: two yeast genes encoding the high-molecular-weight subunits of the cap-binding protein complex (eukaryotic initiation factor 4F) contain an RNA recognition motif-like sequence and carry out an essential function.

The 5' ends of eukaryotic mRNAs are blocked by a cap structure, m7GpppX (where X is any nucleotide). The interaction of the cap structure with a cap-binding protein complex is required for efficient ribosome binding to the mRNA. In Saccharomyces cerevisiae, the cap-binding protein complex is a heterodimer composed of two subunits with molecular masses of 24 (eIF-4E, CDC33) and 150 (p150) kDa. p150 is presumed to be the yeast homolog of the p220 component of mammalian eIF-4F. In this report, we describe the isolation of yeast gene TIF4631, which encodes p150, and a closely related gene, TIF4632. TIF4631 and TIF4632 are 53% identical overall and 80% identical over a 320-amino-acid stretch in their carboxy-terminal halves. Both proteins contain sequences resembling the RNA recognition motif and auxiliary domains that are characteristic of a large family of RNA-binding proteins. tif4631-disrupted strains exhibited a slow-growth, cold-sensitive phenotype, while disruption of TIF4632 failed to show any phenotype under the conditions assayed. Double gene disruption engendered lethality, suggesting that the two genes are functionally homologous and demonstrating that at least one of them is essential for viability. These data are consistent with a critical role for the high-molecular-weight subunit of putative yeast eIF-4F in translation. Sequence comparison of TIF4631, TIF4632, and the human eIF-4F p220 subunit revealed significant stretches of homology. We have thus cloned two yeast homologs of mammalian p220.

Amino Acid Sequence↗
PubMed1992

Interactions of the eIF-4F subunits in the yeast Saccharomyces cerevisiae.

Recognition of the cap structure at the 5' end of mRNA is one of the first events in initiation of eukaryotic translation. This step is mediated by the translation initiation factor 4F (eIF-4F). In mammalian cells this factor is composed of the cap-binding protein eIF-4E, eIF-4A, and a 220-kDa polypeptide. In yeast Saccharomyces cerevisiae, eIF-4E is found associated with a 150-kDa protein (p150) and a 20-kDa protein (p20). The resulting protein complex is proposed to represent yeast eIF-4F. To study the functions of p150 and p20 and their interaction with eIF-4E, we disrupted the genes encoding p150 and p20 and analyzed the effects on protein complex formation and cell viability. Yeast cells with single and double disruptions of the genes encoding p150 and p20 are viable, but p150 single and p150/p20 double disruptions show a slow growth phenotype. Gel chromatography and immunoadsorption experiments with a monoclonal anti-eIF-4E antibody coupled to protein G-Sepharose show that both p150 and p20 bind independently of each other to eIF-4E.

Antibodies, Monoclonal↗
PubMed1992

Ligands induce conformational changes in the carboxyl-terminus of progesterone receptors which are detected by a site-directed antipeptide monoclonal antibody.

We have prepared a monoclonal antibody, C-262, to a synthetic peptide that contains the carboxy-terminal 14 amino acids from progesterone receptors (PR). This sequence is 100% conserved in all species of PRs that have been cloned to date, suggesting that this antibody will recognize all mammalian and avian PR. The C-262 antibody recognizes both native and denatured forms of the receptor. However, it does not recognize PR when they are bound to the hormone agonists progesterone or R5020. Surprisingly the antibody does recognize PR when they are bound to the steroid antagonist RU486. This suggests that progestin agonists induce a conformational change in the receptor that occludes the C-262 epitope in the carboxyl-terminus, whereas unliganded receptors and receptors bound with RU486 assume distinct conformations that leaves the C-terminal tail accessible to the C-262 antibody.

Amino Acid Sequence↗
PubMed1991

Characterization and functional properties of the A and B forms of human progesterone receptors synthesized in a baculovirus system.

Human progesterone receptors (PR) were overexpressed in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus system. Recombinant viruses were constructed that produced either full-length A (94K) or B (120K) forms of human PR, and each was expressed as a functional protein. Steroid and DNA binding activities were found to be indistinguishable from that of endogenous human PR in T47D breast cancer cells. Moreover, as analyzed by gel-mobility shift, recombinant PR-A and PR-B each bound to specific progesterone response elements in a strictly hormone-dependent manner. Native receptors expressed in Sf9 cells also exhibited structural properties similar to that of endogenous PR. Cytosolic PR (PR-A or PR-B), prepared in low salt buffer, sedimented on density gradients as an 8S oligomeric complex that was converted largely to 4S by treatment with 0.4 M NaCl. Immune isolation of the 8S cytosol PR complex and analysis of protein composition revealed the presence of two specific copurifying proteins of approximately 90K and 70K. The 90-K component was identified immunologically as heat shock protein 90. The 70-K component was not identified but is likely to be the insect equivalent of heat shock protein 70. Immune isolation of PR from Sf9 cells metabolically labeled with [32Pi], revealed that expressed PR was capable of being phosphorylated in insect cells. Hormone addition to Sf9 cells, however, did not stimulate the same increase in PR phosphorylation or upshift in mobility on sodium dodecyl sulfate gels that occurs with endogenous receptors in T47D cells. Thus some, but not all, phosphorylations occur with human PR expressed in Sf9 cells. These phosphorylation data, together with the fact that expressed PR required hormone for DNA binding, indicate that the hormone-dependent phosphorylation step responsible for PR upshifts on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is not required for receptor binding to DNA. The baculovirus expression system, therefore, may prove valuable in dissecting the functional role(s) for both hormone-dependent and hormone-independent PR phosphorylation.

Animals↗
PubMed1990

Translation initiation factor-dependent extracts from Saccharomyces cerevisiae.

Translation initiation factor 4A- and 4E-dependent extracts were developed from Saccharomyces cerevisiae and used to study factor requirements for translation of individual mRNAs in vitro. Whereas all mRNAs tested required eIF-4A, mRNAs devoid of secondary structure in their 5' untranslated region did not require exogenous eIF-4E for translation. The latter included alfalfa mosaic virus RNA4, mRNA containing the untranslated region of tobacco mosaic virus RNA and mRNA containing part of the untranslated region of poliovirus RNA. Furthermore, initiation of translation on mRNAs containing part of the untranslated region of poliovirus RNA is most likely internal.

Animals↗
PubMed1990

The 5'-leader sequence of tobacco mosaic virus RNA mediates initiation-factor-4E-independent, but still initiation-factor-4A-dependent translation in yeast extracts.

Messenger RNAs encoding chloramphenicol acetyltransferase (CAT) with or without the 5'-leader sequence of tobacco mosaic virus (TMV) RNA were synthesized in vitro and translated in Saccharomyces cerevisiae extracts dependent on eukaryotic initiation factors eIF-4E or eIF-4A. The 5'-leader sequence of TMV RNA renders translation of CAT mRNA eIF-4E-independent but still 4A-dependent.

Chloramphenicol O-Acetyltransferase↗
PubMed1990

Identification of an Onchocerca volvulus cDNA encoding a low-molecular-weight antigen uniquely recognized by onchocerciasis patient sera.

The primary structure of an immunodominant antigen of the filarial parasite, Onchocerca volvulus was deduced from cDNA sequence analysis. Using affinity-purified antibody from onchocerciasis patients from West Africa, we have isolated a cDNA clone from a lambda gt11 cDNA expression library derived from microfilariae-producing female O. volvulus. The open reading frame encodes 152 amino acids, and the deduced sequence predicts a Mr of 16,850 (consistent with the apparent Mr of 18,000 of the immunoprecipitated in vitro translated product). The primary translation product contains a putative signal peptide of 16 amino acids. The mRNA coding for this antigen has an estimated size of 950 nucleotides. Furthermore, immunoelectron microscopy established that the antigen encoded by this clone is present in the hypodermis, the cuticle, and in the uterus of the filarial worms. Since this antigen is recognized exclusively by sera from onchocerciasis patients, and not by other sera from patients infected by other filarial parasites, it may prove to be an especially valuable tool for improving the specific diagnosis of onchocerciasis.

Amino Acid Sequence↗
PubMed1990

What makes a stopping rule sexist?

"Parents' decisions to have children are modeled by a simple stopping rule that describes the probability of having another child as a function of the number of boys and girls already born to the parents. Because the stopping rule depends on the sex of the offspring, the rule may introduce a correlation between sex of offspring and the number of siblings the offspring has. When this is coupled with a correlation between number of siblings and well-being, a correlation between sex and well-being may emerge despite equal treatment of the two sexes within each family. The author provides sufficient conditions on a stopping rule for it to be sexist in the sense that the average well-being of one sex is higher than that of the other sex." (SUMMARY IN FRE)

Behavior↗
PubMed1990

Monte Carlo simulation of HIV infection in an intravenous drug user community.

Intravenous drug users are crucial to the understanding and control of human immunodeficiency virus (HIV) transmission. We have developed a population-based simulation of a community of intravenous drug users. This model was implemented using Monte Carlo techniques, which permit great flexibility in creating realistic social structures, to describe the needle-sharing network of drug users. We present the baseline behavior of this model in a generic community and demonstrate the model's utility for assessing public health interventions. Our early results demonstrate the powerful effects of social networks on HIV transmission and the importance of prevalence levels in assessing the effectiveness of interventions in the drug-injecting community.

Equipment Contamination↗
PubMed1989

Altered mRNA cap recognition activity of initiation factor 4E in the yeast cell cycle division mutant cdc33.

The mutation in the S. cerevisiae cell cycle division mutant cdc33 consists of a single G to A transition in the open reading frame encoding translation initiation factor 4E (eIF-4E). This leads to the substitution of glycine 113 by aspartic acid close to tryptophane 115 in the protein. This mutation reduces cap binding activity of eIF-4E as measured by binding of eIF-4E to m7GDP agarose columns and slows down overall protein synthesis at the non-permissive temperature. Comparison of the cdc33 mutation with other mutations affecting eIF-4E function supports the view that tryptophane residues and their flanking regions are involved in cap binding activity of eIF-4E.

Amino Acid Sequence↗
PubMed1989

A mammalian translation initiation factor can substitute for its yeast homologue in vivo.

The translation initiation factor 4E (eIF-4E) is involved in the recognition of the cap structure at the 5' end of eukaryotic mRNA and facilitates ribosome binding. Subsequently, additional initiation factors mediate ribosomal scanning of mRNA and initiator AUG recognition (Shatkin, A. J. (1985) Cell 40, 223-224; Rhoads, R. E. (1988) Trends Biochem. Sci. 13, 52-56; Edery, I., Pelletier, J., and Sonenberg, N. (1987) in Translational Regulation of Gene Expression (Ilan, J., ed) pp. 335-366, Plenum Publishing Corp., New York). We show here that initiation factor 4E is functionally conserved between the unicellular eukaryote Saccharomyces cerevisiae and mammals. Although the amino acid identity of the factors from both species is limited to only 33%, mouse eIF-4E can substitute for yeast eIF-4E in vivo without major effects on cell viability, growth, and mating. This finding provides a starting point for new experimental strategies to investigate the structure-function relationship of eukaryotic translation initiation factor eIF-4E.

Amino Acid Sequence↗
PubMed1989

Identification and characterization of cap-binding proteins from yeast.

Photochemical cross-linking of Saccharomyces cerevisiae ribosomal salt wash preparations to cap-labeled mRNA reveals, in addition to the previously characterized 24-kDa cap-binding protein (eIF-4E), the presence of two novel cap-binding proteins (CBPs) of apparent molecular masses of 96 and 150 kDa. Cross-linking of the 96-kDa CBP was found to occur spontaneously without UV light induction. Based on the ATP/Mg2+ requirements, the three CBPs can be subdivided into two classes: 1) ATP/Mg2+ independent (24- and 150 kDa) and 2) Mg2+ dependent (96 kDa). The co-purification of the 24- and 150-kDa CBPs through several different chromatographic steps is consistent with the existence of a yeast CBP complex, possibly analogous to mammalian eIF-4F.

Adenine Nucleotides↗
PubMed1989

Circular dichroism and fluorescence studies on five mutant forms of protein synthesis initiation factor eIF-4E, from the yeast Saccharomyces cerevisiae.

CD studies have shown that five tryptophan to phenylalanine (W----F) mutants of eukaryotic initiation factor-4E (eIF-4E) contain low amounts of alpha-helix, the main elements of secondary structure being beta-sheets/turns and aperiodic regions. Interactions with the cap analog m7GpppG are accompanied by changes in overall secondary structure which include reductions, and in one case an increase in alpha-helix content, as well as increases in total beta-structure (3 mutant forms) and decreases in total beta-structure (2 mutant forms). These changes may also involve more significant perturbations of localized regions containing phenylalanine residues either involved in nucleotide binding, or close to the nucleotide-binding site. Measurements of intrinsic Trp fluorescence have shown different quantum yields and reduced m7GpppG-induced quenching (with one exception). Acrylamide quenching studies yielded similar parameters for 4 of the mutants but 1 form displayed significantly reduced values. Melting experiments showed that the Trp fluorescence of 4 of the mutants decreased as the temperature was increased, this effect being reduced in 3 cases in the presence of m7GpppG. W 58 F showed an increase in fluorescence as the temperature was raised and this effect was accentuated in the presence of nucleotide. A preliminary attempt has been made to correlate the spectroscopic data with the known biological importance of the individual Trp residues.

Acrylamides↗
PubMed1989

Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system.

The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The mutagenized gene was reintroduced on a plasmid into S. cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter. Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium. Under these conditions, the phenotype conferred upon the cells by the mutated eIF-4E gene became apparent. Temperature-sensitive S. cerevisiae strains were identified by replica plating. The properties of one strain, 4-2, were further analyzed. Strain 4-2 has two point mutations in the eIF-4E gene. Upon incubation at 37 degrees C, incorporation of [35S]methionine was reduced to 15% of the wild-type level. Cell-free translation systems derived from strain 4-2 were dependent on exogenous eIF-4E for efficient translation of certain mRNAs, and this dependence was enhanced by preincubation of the extract at 37 degrees C. Not all mRNAs tested required exogenous eIF-4E for translation.

Amino Acid Sequence↗
PubMed1988

High-level synthesis in Escherichia coli of functional cap-binding eukaryotic initiation factor eIF-4E and affinity purification using a simplified cap-analog resin.

Numerous studies have established the important role that eukaryotic initiation factor-4E (eIF-4E) plays during protein biosynthesis. However, biochemical characterization of eIF-4E has proved difficult, mainly because of its low abundance in cells. To facilitate studies on eIF-4E, we have overexpressed Saccharomyces cerevisiae eIF-4E in Escherichia coli. The isolation of eIF-4E was simplified by using a cap-analog affinity matrix (agarose resin) that is considerably less demanding to prepare than those previously reported. We describe a simple and rapid purification scheme that can yield 2-5 micrograms of a homogenous and active preparation of eIF-4E from 1 ml of E. coli culture. E. coli-expressed eIF-4E is active as determined by its ability to bind the cap structure. The results demonstrate that the cap-binding activity of eIF-4E is not dependent on the presence of other proteins that are present at low levels in eIF-4E preparations isolated from eukaryotic cells.

Base Sequence↗
PubMed1988

Site-directed mutagenesis of the tryptophan residues in yeast eukaryotic initiation factor 4E. Effects on cap binding activity.

Initiation factor 4E is a 24-kilodalton polypeptide that binds specifically to the 5' cap structure of eukaryotic mRNAs. Sequence analysis of cDNA clones of initiation factor 4E from several species revealed a high tryptophan content (8 residues). Strikingly, all tryptophans are conserved evolutionarily in number and position between yeast and mammals. Here we show, using site-directed mutagenesis, that two of the tryptophans (those referred to as numbers 1 and 8) are absolutely required for the cap binding activity of an Escherichia coli expressed initiation factor 4E.

Amino Acid Sequence↗