Biomedical subjects

F Sherman

Publications and source records attributed to F Sherman.

At least 55 records · Page 3Linked to original sources

PubMed1997

Variation in assimilating functions occurs in spontaneous Candida albicans mutants having chromosomal alterations.

In this study, four clinical isolates and over 100 colony morphology mutants, previously derived spontaneously from strain 3153A during growth on glucose medium, were examined for their utilization of 21 carbon and 3 nitrogen sources at various growth temperatures. The results demonstrated extensive variability in the pattern of assimilation among the mutants and strains, including both the gain and loss of assimilating functions. The persistent alterations in assimilation patterns observed in sequentially produced subclones illustrated an extensive ability of C. albicans populations to constantly produce new combinations of assimilating functions. The variability among spontaneous mutants derived from a single strain explains the well documented variability among natural isolates. From these results we established a relationship between the previously documented broad spectrum of spontaneous chromosomal aberrations in these mutants to the expression of genes controlling the utilization of alternative carbon and nitrogen sources. The existence of cryptic genes, responsible for growth on alternative substrates, was previously deduced from the analysis of other mutants obtained as a response to the restrictive condition on media containing non-assimilating carbon sources. Thus, mutants with altered assimilation functions can arise either on glucose medium or by selection on restricted media. Extensive differences between the patterns of chromosomal aberrations and the distribution of correlated phenotypes in the two groups of mutants indicated that the same phenotypes may be produced by two different mechanisms involving the same or different genes.

Amino Acids↗

PubMed1997

Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis.

The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.

Alleles↗

PubMed1996

Degradation of CYC1 mRNA in the yeast Saccharomyces cerevisiae does not require translation.

Several studies have indicated that degradation of certain mRNAs is tightly coupled to their translation, whereas, in contrast, other observations suggested that translation can be inhibited without changing the stability of the mRNA. We have addressed this question with the use of altered CYC1 alleles, which encode iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. The cyc1-1249 mRNA, which lacks all in-frame and out-of-frame AUG triplets, was as stable as the normal mRNA. This finding established that translation is not required for the degradation of CYC1 mRNAs. Furthermore, poly(G)18 tracks were introduced within the CYC1 mRNA translated regions to block exonuclease degradation. The recovery of 3' fragments revealed that the translatable and the AUG-deficient mRNAs are both degraded 5'-->3'. Also, the increased stability of CYC1 mRNAs in xrn1-delta strains lacking Xrn1p, the major 5'-->3' exonuclease, established that the normal and AUG-deficient mRNAs are degraded by the same pathway. In addition, deadenylylation, which activates the action of Xrn1p, occurred at equivalent rates in both normal and AUG-deficient mRNAs. We conclude that translation is not required for the normal degradation of CYC1 mRNAs, and that translatable and untranslated mRNAs are degraded by the same pathway.

Base Sequence↗

PubMed1996

Side chain packing of the N- and C-terminal helices plays a critical role in the kinetics of cytochrome c folding.

The pairing of two alpha-helices at opposite ends of the chain is a highly conserved structural motif found throughout the cytochrome c family of proteins. It has previously been shown that association of the N- and C-terminal helices is a critical early event in the folding process of horse cytochrome c and is responsible for the formation of a partially folded intermediate (INC). In order to gain further insight into the structural and energetic basis of helix packing interactions and their role in folding, we prepared a series of horse cytochrome c variants in which Leu94, a critical residue at the helix contact site, was replaced by Ile, Val, or Ala. The Ile and Val substitutions resulted in minor changes in the stability of the native state, indicating that conservative mutations can be accommodated at the helix interface with only minor structural perturbations. In contrast, the L94A mutation resulted in a 3.5 kcal/mol decrease in unfolding free energy, suggesting that the smaller Ala side chain causes severe packing defects at the helix interface. The effect of these mutations on the kinetics of folding and unfolding as a function of denaturant concentration was studied by a systematic series of stopped-flow fluorescence measurements. The proteins with Leu, Ile, or Val at position 94 exhibit a major unresolved fluorescence change during the 1-ms dead time of the stopped-flow refolding measurements, while this effect is less pronounced in L94A, indicating that the rapid formation of a compact state (IC) with largely quenched Trp59 fluorescence is favored by a large hydrophobic side chain at the helix-helix interface. Despite their small effects on overall stability, the L94I and L94V mutations result in a substantial reduction in the relative amplitude of the fastest observable folding phase (formation of INC) consistent with a strong decrease in the population of INC compared to the wild-type protein. This effect is amplified in the case of the destabilizing L94A variant, which exhibits slower folding kinetics and negligible accumulation of INC. Whereas the presence of a large hydrophobic side chain at position 94 is sufficient for the stabilization of IC, the subsequent partially folded intermediate, INC, is stabilized by specific interactions that are responsible for the proper packing of the two alpha-helices.

Animals↗

PubMed1996

Modulated growth of Saccharomyces cerevisiae by altering the driving force of the reactions of cytochrome c: Marcus' theory in vitro and in vivo.

According to Marcus' theory, rates of electron transfer reactions depend parabolically on the free energy of reaction. Amino acid replacements in the electron transport protein cytochrome c produced a series of proteins which changed the free energy of reaction for cytochrome c in oxidative phosphorylation. This study shows that Marcus' theory of electron transfer can be applied to the reactions of redox-altered cytochromes c with cytochrome c1 both in vitro and in vivo. In vitro, isolation of physiologically relevant partners of cytochrome c suggests that a change in free energy of reaction of cytochrome c changes the rate of electron transfer with cytochrome bc1 complex as would be predicted by Marcus' theory of electron transfer. Furthermore, the reactivity pattern observed in vitro is paralleled in in vivo studies. In vivo the rates of growth of Saccharomyces cerevisiae, in which these alternatives have been incorporated, also are consistent with the change in free energy of the reactions of cytochrome c with cytochrome bc1 complex. This study suggests that Marcus' theory of electron transport can predict rates not only in vitro, in isolated protein-protein systems, but also in vivo, where the relative growth rates of yeast may be predicted from the in vitro results.

Cytochrome c Group↗

PubMed1996

Sequence requirements for mitochondrial import of yeast cytochrome c.

Apocytochrome c is synthesized in the cytoplasm, transported to the mitochondrial intermembrane space, and subsequently covalently attached to heme in a reaction catalyzed by the enzyme cytochrome c heme lyase. We have investigated the amino acid sequences in cytochrome c which are required for mitochondrial import, using a systematic series of site-directed alterations of the CYC7-H3 gene which encodes iso-2-cytochrome c in the yeast Saccharomyces cerevisiae. Import of the altered apocytochromes c was assayed in yeast strains that overexpressed cytochrome c heme lyase. Under these conditions, there was efficient mitochondrial accumulation of forms of apocytochrome c which are incapable of having heme covalently attached. In fact, all apocytochromes c containing deletions located to the carboxyl-terminal side of His27 efficiently accumulated in the mitochondria of strains overexpressing heme lyase, even though all but one of these deletion-containing proteins were incapable of heme attachment. A minimum length of polypeptide chain at the extreme amino terminus of cytochrome c, rather than any specific sequence element in this region, appears to be required for efficient mitochondrial import. Certain amino acid substitutions in the region extending from Gly15 to Leu18, at residue Phe19 and at residue His27, lead to reduced mitochondrial import of apocytochrome c, resulting from stalling of the altered apocytochrome c in partially imported states.

Amino Acid Sequence↗

PubMed1996

Review: the Cct eukaryotic chaperonin subunits of Saccharomyces cerevisiae and other yeasts.

All eight of the CCT1-CCT8 genes encoding the subunits of the Cct chaperonin complex in Saccharomyces cerevisiae have been identified, including three that were uncovered by the systematic sequencing of the yeast genome. Although most of the properties of the eukaryotic Cct chaperonin have been elucidated with mammalian systems in vitro, studies with S. cerevisiae conditional mutants revealed that Cct is required for assembly of microtubules and actin in vivo. Cct subunits from the other yeasts, Candida albicans and Schizosaccharomyces pombe, also have been identified from partial and complete DNA sequencing of genes. Cct8p from C. albicans, the only other completely sequenced Cct protein from a fungal species other than S. cerevisiae, is 72% and 61% similar to the S. cerevisiae and mouse Cct8 proteins, respectively.

Actins↗

PubMed1996

3'-end-forming signals of yeast mRNA.

The signals required for forming 3'-ends of mRNAs from the yeast Saccharomyces cerevisiae differ from the corresponding signals of higher eukaryotes. Yeast signals consist of three elements: (1) the efficiency element, which enhances the efficiency of downstream positioning elements; (2) the positioning element, which positions the poly(A) site; and (3) the actual poly(A) site. These three elements are not only necessary, but also sufficient for mRNA 3'-end formation in yeast.

Animals↗

PubMed1996

mRNA sequences influencing translation and the selection of AUG initiator codons in the yeast Saccharomyces cerevisiae.

The secondary structure and sequences influencing the expression and selection of the AUG initiator codon in the yeast Saccharomyces cerevisiae were investigated with two fused genes, which were composed of either the CYC7 or CYC1 leader regions, respectively, linked to the lacZ coding region. In addition, the strains contained the upf1-delta disruption, which stabilized mRNAs that had premature termination codons, resulting in wild-type levels. The following major conclusions were reached by measuring beta-galactosidase activities in yeast strains having integrated single copies of the fused genes with various alterations in the 89 and 38 nucleotide-long untranslated CYC7 and CYC1 leader regions, respectively. The leader region adjacent to the AUG initiator codon was dispensable, but the nucleotide preceding the AUG initiator at position -3 modified the efficiency of translation by less than twofold, exhibiting an order of preference A > G > C > U. Upstream out-of-frame AUG triplets diminished initiation at the normal site, from essentially complete inhibition to approximately 50% inhibition, depending on the position of the upstream AUG triplet and on the context (-3 position nucleotides) of the two AUG triplets. In this regard, complete inhibition occurred when the upstream and downstream AUG triplets were closer together, and when the upstream and downstream AUG triplets had, respectively, optimal and suboptimal contexts. Thus, leaky scanning occurs in yeast, similar to its occurrence in higher eukaryotes. In contrast, termination codons between two AUG triplets causes reinitiation at the downstream AUG in higher eukaryotes, but not generally in yeast. Our results and the results of others with GCN4 mRNA and its derivatives indicate that reinitiation is not a general phenomenon in yeast, and that special sequences are required.

Base Sequence↗

PubMed1996

Signals sufficient for 3'-end formation of yeast mRNA.

The following three elements were previously shown to be required for 3'-end formation of mRNA in the yeast Saccharomyces cerevisiae: (i) the efficiency element TATATA or related sequences, which function by enhancing the efficiency of downstream positioning elements; (ii) the positioning element AATAAA or related sequences, which position the poly(A) site; and (iii) the actual poly(A) site, which is usually Py(A)n. In this study, we synthesized a 39-pb poly(A) signal that contained the optimum sequences of these three elements. By inserting the synthetic 3'-end-forming signal into various positions of a CYC1-lacZ fusion gene, we showed that truncated transcripts of the expected sizes were generated. Furthermore, the poly(A) sites of the truncated transcripts were mapped to the expected poly(A) site within the synthetic signal. Our findings establish that the three elements are not only necessary but also sufficient for mRNA 3'-end formation in S. cerevisiae.

Base Sequence↗

PubMed1995

The BALB/c mouse B-cell response to pigeon cytochrome c initiates as a heteroclitic response specific for the self antigen mouse cytochrome c.

Direct evidence is presented in support of the longstanding but unproven hypothesis that B lymphocytes specific for self antigens (Ags) can be used in the immune response to foreign Ags. We show that the B cells in BALB/c mic responding early to pigeon cytochrome c (CYT) produce antibodies that recognize and bind the major antigenic site on mouse CYT with greater affinity than they bind pigeon CYT i.e., they are heteroclitic for the self Ag. Furthermore, these B cells express the same combination of immunoglobulin variable region (V) genes that are known to be used in B-cell recognition of mouse CYT. Over time, the response to pigeon CYT becomes more specific for the foreign Ag through the recruitment of B cells expressing different combinations of V genes and, possibly, somatic mutation of the mouse CYT specific B cells from early in the response. Cross-recognition of pigeon CYT by mouse CYT-specific B cells results from the sharing of critical amino acid residues by the two Ags. Although B-cell recognition of the self Ag, mouse CYT, is very specific, which limits the extent to which foreign Ags can cross-activate the autoreactive B cells, it is possible that polyreactive B cells to other self Ags may be used more frequently in response to foreign Ags.

Amino Acid Sequence↗

PubMed1995

Enhanced stability in vivo of a thermodynamically stable mutant form of yeast iso-1-cytochrome c.

Previous work has established that the N57I amino acid replacement in iso-1-cytochrome c from the yeast Saccharomyces cerevisiae causes an unprecedented increase in thermodynamic stability of the protein in vitro, whereas the N57G replacement diminishes stability. Spectrophotometric measurements of intact cells revealed that the N57I iso-1-cytochrome c is present at higher than normal levels in vivo. Although iso-1-cytochrome c turnover is negligible during aerobic growth, transfer of fully derepressed, aerobically grown cells to anaerobic growth conditions leads to reduction in the levels of all of the cytochromes. Pulse-chase experiments carried out under these anaerobic conditions demonstrated that the N57I iso-1-cytochrome c has a longer half-life than the normal protein. This is the first report of enhanced stability in vivo of a mutant form of a protein that has an enhanced thermodynamic stability in vitro. Although the N57I protein concentration is higher than the normal level, reduced growth in lactate medium indicated that the specific activity of this iso-1-cytochrome c in vivo is diminished relative to wild-type. On the other hand, the level of the thermodynamically labile N57G iso-1-cytochrome c was below normal. The in vivo levels of the N57I and N57G iso-1-cytochrome c suggest that proteins in the mitochondrial intermembrane space can be subjected to degradation, and that this degradation may play a role in controlling their normal levels.

Aerobiosis↗

PubMed1995

Degradation of cytochrome oxidase subunits in mutants of yeast lacking cytochrome c and suppression of the degradation by mutation of yme1.

We have confirmed by spectral analysis that cytochrome oxidase is not present in strains of the yeast Saccharomyces cerevisiae having a primary deficiency in cytochrome c, and we have demonstrated by immunological procedures that such strains lack the mitochondrially encoded subunits I, II, and III of cytochrome oxidase. Furthermore, pulse-chase experiments demonstrated that subunit II is rapidly degraded in vivo. This degradation can be at least partially suppressed by disruption of the nuclear gene YME1, which encodes a putative ATP-Zn(2+)-dependent protease. We suggest that the cytochrome oxidase subunits are not properly assembled in the absence of cytochrome c, and that Yme1 and possibly other proteases degrade the unassembled mitochondrial-encoded subunits of cytochrome oxidase.

ATP-Dependent Proteases↗

PubMed1995

Stabilizing amino acid replacements at position 52 in yeast iso-1-cytochrome c: in vivo and in vitro effects.

Position 52 of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae was systematically replaced with all possible amino acids to investigate the molecular basis of the "global suppressor" activity for the N52I mutation. Isogenic strains containing the variant proteins were made with a mixed oligonucleotide-directed mutagenesis technique in vivo. A relationship between thermodynamic protein stability and cellular protein levels was established by comparing direct spectroscopic measurements of cytochrome c levels in vivo with the thermodynamic parameters from guanidine hydrochloride denaturation and microcalorimetric measurements. Reversible denaturation data show that, within a large group of amino acid side chains, the thermodynamic stabilization at position 52 is related to the hydrophobicity of the side chain at that site.

Amino Acid Sequence↗

PubMed1995

Site specific combinations of stabilizing and destabilizing amino acid replacements in yeast cytochrome c: in vivo and in vitro effects.

Oligonucleotide-directed mutagenesis in vivo was used to create destabilizing site specific changes at position 6 in iso-1-cytochrome c in Saccharomyces cerevisiae. These changes were made in combination with the stabilizing N52I substitution. The resulting proteins showed that a variety of forces and interactions are responsible for these destabilizations. Increasing side chain size was the strongest predictor of decreases in cytochrome c levels in vivo. With intermediate size replacements, increasing hydrophobicity correlated with the proteins' thermostability. Some differences in protein levels in vivo could not be explained by side chain size and hydrophobicity alone. Therefore, specific interactions of individual amino acids may also be involved. The N52I-stabilizing mutation tended to increase the protein levels to the same degree relative to the amino acid at position 6. These stabilized cytochromes had an increased specific activity when compared to the series with the original N52. Strains with these altered cytochromes c showed temperature sensitivities for protein levels and function. Thermodynamic measurements in vivo of the WT (C102A), N52I, G6A, G6A N52I, and G6S N52I correlated with the in vivo data. The variant G6A N52I showed additivity (Wells, 1990) of the Cm's and delta delta G's of unfolding for guanidine hydrochloride denaturation.

Amino Acid Sequence↗

PubMed1995

Redundant 3' end-forming signals for the yeast CYC1 mRNA.

The cyc1-512 mutation is a 38-bp deletion in the 3' untranslated region of the CYC1 gene, which encodes iso-1-cytochrome c in Saccharomyces cerevisiae. This deletion caused a 90% reduction in the levels of the CYC1 mRNA and protein because of the absence of the normal 3' end-forming signal. Although the 3' end-forming signal was not defined by previous analyses, we report that concomitant alteration by base-pair substitution of three 3' end-forming signals within and adjacent to the 38-bp region produced the same phenotype as the cyc1-512 mutation. Furthermore, these signals appear to be related to the previously identified 3' end-forming signal TATATA. A computer analysis revealed that TATATA and related sequences were present in the majority of 3' untranslated regions of yeast genes. Although TATATA may be the strongest and most frequently used signal in yeast genes, the CYC1+ gene concomitantly employed the weaker signals TT-TATA, TATGTT, and TATTTA, resulting in a strong signal.

Base Sequence↗

PubMed1995

Diminished degradation of yeast cytochrome c by interactions with its physiological partners.

The level and structure of yeast iso-1-cytochrome c and iso-2-cytochrome c, encoded by the nuclear genes CYC1 and CYC7, respectively, are normally not altered in rho- mutants, which completely lack the cytochromes a.a3 subunits and cytochrome b that are encoded by mitochondrial DNA. In contrast, iso-cytochromes c containing the amino acid change Thr-78-->Ile (T78I) were observed at the normal or near-normal wild-type level in rho+ strains but were completely absent in rho- mutants. We have demonstrated with the "global" suppressor mutation Asn-52-->Ile and by pulse-chase labeling that the T78I iso-1-cytochrome c undergoes rapid cellular degradation in rho- mutants. Furthermore, specific mutations revealed that the deficiency of T78I iso-1 cytochrome c can be caused by the lack of cytochrome a.a3 or cytochrome c1, but not by the lack of cytochrome b. Thus, this and certain other, but not all, labile forms of cytochrome c are protected from degradation by the interaction with its physiological partners.

Alleles↗

PubMed1995

Structural and functional effects of multiple mutations at distal sites in cytochrome c.

Multiple mutations at distally located sites have been introduced into yeast iso-1 cytochrome c to determine the contributions of three amino acids to the structural and functional properties of this protein. The mutant proteins, for which high-resolution structures were determined, included all possible combinations of the substitutions Arg38Ala, Asn52Ile, and Phe82Ser. Arg38, Asn52, and Phe82 are all conserved in the primary sequences of eukaryotic cytochromes c and have been shown to significantly affect several properties of these proteins including protein stability, heme reduction potential, and oxidation state dependent conformational changes. The present studies show that the structural consequences of each amino acid substitution in combinatorial mutant proteins were similar to those observed in the related single-mutant proteins, and therefore no synergistic effect between mutation sites was observed for this feature. With respect to protein stability, the effect of individual mutations can be understood from the structural changes observed for each. It is found that stability effects of the three mutation sites are independent and cumulative in multiple-mutant proteins. This reflects the independent nature of the structural changes induced at the three distally located mutation sites. In terms of heme reduction potential two effects are observed. For substitution of Phe82 by serine, the mechanism by which reduction potential is lowered is different from that occurring at either the Arg38 or the Asn52 site and is independent of residue replacements at these latter two positions. For Arg38 and Asn52, overlapping interactions lead to a higher reduction potential than expected from a strict additive effect of substitutions at these residues. This appears to arise from interaction of these two amino acids with a common heme element, namely, the heme propionate A group. The present results underscore the difficulty of predicting synergistic effects of multiple mutations within a protein.

Crystallography, X-Ray↗