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F Sherman

Publications and source records attributed to F Sherman.

At least 91 records · Page 5Linked to original sources
PubMed1992

Parameters affecting the frequencies of transformation and co-transformation with synthetic oligonucleotides in yeast.

Factors influencing the direct transformation of the yeast Saccharomyces cerevisiae with synthetic oligonucleotides were investigated by selecting for cyc1 transformants that contained at least partially functional iso-1-cytochrome c. Approximately 3 x 10(4) transformants, constituting 0.1% of the cells, were obtained by using 1 mg of oligonucleotide in the reaction mixture. Carrier, such as heterogeneous oligonucleotides, enhanced transformation frequencies. Transformation frequencies were dramatically reduced if the oligonucleotides had a large number of mismatches or had terminally located mismatches. Transformation with oligonucleotides, but not with linearized double-strand plasmid, was efficient in a rad52- strain, suggesting that the pathway for transformation with oligonucleotides is different from that with linearized double-strand plasmid. We describe a procedure of co-transformation with two oligonucleotides, one correcting the cyc1 defect of the target allele in the host strain, and the other producing a desired amino acid alteration elsewhere in the iso-1-cytochrome c molecule; approximately 20% of the transformants obtained by co-transformation contained these desired second alterations.

Amino Acid Sequence↗
PubMed1992

Strand-specificity in the transformation of yeast with synthetic oligonucleotides.

Cyc1 mutants of the yeast Saccharomyces cerevisiae were directly transformed with both sense and antisense oligonucleotides to examine the involvement of the two genomic DNA strands in transformation. Sense oligonucleotides yielded approximately 20-fold more transformants than antisense oligonucleotides. This differential effect was observed with oligonucleotides designed to make alterations at six different sites along the gene and was independent of the oligonucleotide sequence and length, number of mismatches and the host strain. Competition studies showed that antisense oligonucleotides did not inhibit transformation. Although the mechanism for this strand specificity is unknown, this difference was maintained even when CYC1 transcription was diminished to approximately 2% of the normal level.

Alleles↗
PubMed1992

cis- and trans-acting suppressors of a translation initiation defect at the cyc1 locus of Saccharomyces cerevisiae.

The cyc1-362 mutant of Saccharomyces cerevisiae is deficient in iso-1-cytochrome c as a consequence of an aberrant ATG codon that initiates a short open reading frame (uORF) in the cyc1 transcribed leader region. We have isolated and characterized functional revertants of cyc1-362 in an effort to define cis- and trans-acting factors that can suppress the effect of the uORF. Genetic and DNA sequence analyses have defined three classes of revertants: (i) those that acquired point mutations in the upstream ATG (uATG), restoring iso-1-cytochrome c to its normal level; (ii) substitution of the normal A residue at position -1 relative to the uATG by either C or T, enhancing iso-1-cytochrome c production from approximately 2% to 6% (C) or 10% (T) of normal, indicating that the nucleotide immediately preceding the initiator codon can affect the efficiency of AUG start codon recognition and that purines are preferred over pyrimidines at this site; and (iii) extragenic suppressors that enhance iso-1-cytochrome c expression to 10-40% of normal while retaining the uATG. These suppressors are represented by five different genes, designated sua1-sua4 and sua6. In contrast to the previously described sua7 and sua8 suppressors, they do not compensate for the uATG by affecting cyc1 transcription start site selection. Potential suppressor mechanisms are discussed.

Amino Acid Sequence↗
PubMed1991

Direct electrochemistry of proteins. Investigations of yeast cytochrome c mutants and their complexes with cytochrome b5.

Direct electrochemistry of site-specific mutants of yeast iso-1-cytochrome c (cyt c) and their complexes with bovine cytochrome b5 (cyt b5) has been investigated at edge-plane pyrolytic graphite (EPG) and bis(4-pyridyl)-disulphide-modified gold electrodes. Structure/function relationships have been investigated with the particular aim of clarifying the factors controlling the interactions of proteins at electrode/electrolyte interfaces and the determinants for direct electrochemistry in ternary protein/protein/electrode adducts, e.g. cyt c/cyt b5/EPG. Investigations of the cyt c mutants alone revealed a variety of electrochemical responses: all the mutants show similar voltammetric reversibility at modified gold electrodes, whereas at EPG electrodes the reversibility follows the order: Asn52Ile-Cys102Thr greater than Cys102Thr greater than Asn52Ala-Cys102Thr. Mid-point potentials follow the order: Arg13Ile (+60 +/- 5 mV vs. standard calomel electrode) greater than Cys102Thr (+40 +/- 5 mV) greater than Lys27Gln (+30 +/- 5 mV) approximately Lys72Asp (+30 +/- 5 mV) greater than Asn52Ala-Cys102Thr (+15 +/- 5 mV) greater than Asn52Ile-Cys102Thr (-10 +/- 5 mV). The structural basis for these differences is briefly discussed. When these mutants are bound to cyt b5, the differences in electrochemical response are greatly enhanced in the ternary cyt c/cyt b5/EPG adducts. A minimal analysis of these differences supports a model of multiple overlapping binding and recognition domains on cyt c which may be finely tuned to allow ternary complex formation so that a single-site variation could modify or abolish direct electrochemistry in the ternary adduct.

Animals↗
PubMed1991

The role of the internal hydrogen bond network in first-order protein electron transfer between Saccharomyces cerevisiae iso-1-cytochrome c and bovine microsomal cytochrome b5.

An internal water molecule (designated WAT166) is found in iso-1-cytochrome c which is part of a redox-state-dependent hydrogen bond network. The position of this water molecule with respect to the polypeptide fold can be altered or even displaced by site-directed mutagenesis leading to structural perturbations and associated changes in redox potential. Using saturation transfer 1H-NMR methods, this study measures changes in the electron transfer reactivity for three variants of yeast iso-1-cytochromes c in which the position of this water molecule is altered. In particular, the reverse electron transfer rate is measured within a complex formed between either wild-type or variant yeast iso-1-cytochromes c and the tryptic fragment of bovine liver microsomal cytochrome b5. For three variants of yeast iso-1-cytochrome c the rate constants measured by saturation transfer are wild-type (Asn52, E0 = 270 mV, kex = 0.3 s-1), Asn52----Ala (E0 = 240 mV, kex = 0.6 s-1), Asn52----Ile (E0 = 220 mV, kex = 1.0 s-1). The first-order rates are compared with that of a fourth variant Phe82----Gly which has been measured previously (E0 = 220 mV, kex = 0.7 s-1). An analysis of the variation in the observed cross exchange rate using Marcus theory shows that these changes can be predicted quantitatively by the shift in redox potential that accompanies mutagenesis. So, although the perturbation of the internal water molecule by mutagenesis alters both the structure and redox potential of cytochrome c, surprisingly it does not significantly influence the intrinsic electron transfer reactivity of the protein. Studies of the activation parameters suggests that a variation of temperature changes both delta G* and also the prefactor. These data are discussed in terms of models involving dynamic molecular recognition between proteins.

Animals↗
PubMed1991

Synthesis and expression of genes encoding tuna, pigeon, and horse cytochromes c in the yeast Saccharomyces cerevisiae.

Genes encoding tuna, pigeon, and horse cytochromes c were constructed with synthetic oligodeoxyribonucleotides having preferred codons and portions of the iso-1-cytochrome c-encoding gene from the yeast Saccharomyces cerevisiae. The genes were ligated into an expression vector, which contains the normal 5'- and 3'-untranslated regions of the yeast iso-1-cytochrome c gene, and were integrated in single copy into the chromosome. Yeast strains were also constructed with multiple integrated copies of the pigeon gene. The heterologous and normal mRNA levels of the single-copy strains were equivalent. Although the N-terminal methionines were completely cleaved in the heterospecific proteins, the levels of trimethylation of Lys72 and acetylation of N-terminal glycines ranged from 39-78% and 10-70%, respectively. Horse cytochrome c was produced at a nearly normal level, whereas the pigeon and tuna cytochromes c were produced at approx. 40% of the normal levels. The levels of the cytochromes c and growth of the mutant yeast strains indicated that the heterospecific cytochromes c had approx. 50% specific activity in vivo.

Acetylation↗
PubMed1991

Enhanced thermodynamic stabilities of yeast iso-1-cytochromes c with amino acid replacements at positions 52 and 102.

We have determined the structures and thermodynamic stabilities of the wild type Asn-52 and unusually thermostable mutant Ile-52 yeast iso-1-cytochromes c (Das, G., Hickey, D. R. McLendon, D., McLendon, G., and Sherman, F. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 496-499). Although both structures were similar, Water-166, buried within the wild type protein, is excluded from the Ile-52 mutant, which substantially reorganizes the local hydrogen bonding. Wild type Cys-102 was replaced with alanine or serine to eliminate dimerization in vitro. The Cys-102 (wild type), Ala-102, and Ser-102 proteins were equally stable, whereas the chemically modified Cys-102-SCH3 was less stable. The order of stability observed with replacements at positions 52 and 102 was as follows: Ile-52 Ala-102 greater than Ala-52 Ala-102 greater than Asn-52 Ala-102 ("normal") greater than Gly-52 Ala-102. No significant stabilization was attributed to potential energy interactions expressed as helix-forming propensities of replacements at position 52. A high correlation between differences in free energy changes and transfer free energies suggests hydrophobic interactions are the main factor for enhancing stability in the Ile-52 mutant. Additional possible contributions to the thermostability of the Ile-52 variant are energetic effects due to packing and hydrogen bonding changes surrounding position 52.

Amino Acids↗
PubMed1991

Distinct cis-acting signals enhance 3' endpoint formation of CYC1 mRNA in the yeast Saccharomyces cerevisiae.

The cyc1-512 mutant of the yeast Saccharomyces cerevisiae contains a 38 bp deletion in the 3' untranslated region of the CYC1 gene, resulting in CYC1 mRNAs that are elongated, presumably labile, and reduced to 10% of the normal level. Analysis with S1 nuclease and a novel PCR procedure revealed that the low amount of cyc1-512 mRNA contained many discrete 3' termini at certain sites, ranging from the wild-type position to over 2000 nucleotides (nt) downstream. The cyc1-512 mRNA deficiency was completely or almost completely restored in eight intragenic revertants that contained six different single and multiple base-pair changes within a 300 bp region downstream from the translation terminator codon. Two of the six different reversions formed the sequence TAG...TATGTA, whereas the other four reversions created the sequences TATATA or TACATA. The positions of these revertant sequences varied, even though they caused an increased use of specific major downstream mRNA 3' endpoints, apparently identical to those seen in the cyc1-512 mRNA. However, several revertants contained minor end points not corresponding to any of the cyc1-512 mRNAs. The capacity of these three signals to form 3' ends was confirmed with sequences constructed by site-directed mutagenesis. We therefore suggest that the production of 3' termini of yeast mRNA may involve at least two functionally distinct elements working in concert. One type of element determines the sites of preferred 3' mRNA termini, as represented by the cyc1-512 termini. The second type of element, which includes TAG...TATGTA and TATATA motifs, operates at a distance to enhance the use of the downstream 3' preferred sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

The influence of site-specificity of single amino acid substitutions on electrophoretic separation of yeast iso-1-cytochrome c.

This study dealt with the ability of non-denaturing gel electrophoresis to separate iso-1-cytochrome c with single amino acid replacements isolated from revertants of various cyc1 nonsense mutants of the yeast Saccharomyces cerevisiae. A total of 28 different iso-1-cytochromes c with single amino acid substitutions of one of seven amino acids at six positions were examined on nondenaturing polyacrylamide gels at pH 4.8. Each of these iso-1-cytochromes c exhibited 1 of 16 distinct electrophoretic mobilities. We could distinguish the majority of iso-1-cytochromes c, even those having the same replacement at different sites and those having different replacements that resulted in the same net charge. These results provide confirmation of the importance of site-specific effects on the electrophoretic mobility, and presumably other properties, of proteins differing in sequence by as little as one amino acid. They demonstrate that nondenaturing electrophoresis is able to separate the majority of, but not all, proteins differing by single amino acids.

Amino Acids↗
PubMed1991

Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae.

Heme is covalently attached to cytochrome c by the enzyme cytochrome c heme lyase. To test whether heme attachment is required for import of cytochrome c into mitochondria in vivo, antibodies to cytochrome c have been used to assay the distributions of apo- and holocytochromes c in the cytoplasm and mitochondria from various strains of the yeast Saccharomyces cerevisiae. Strains lacking heme lyase accumulate apocytochrome c in the cytoplasm. Similar cytoplasmic accumulation is observed for an altered apocytochrome c in which serine residues were substituted for the two cysteine residues that normally serve as sites of heme attachment, even in the presence of normal levels of heme lyase. However, detectable amounts of this altered apocytochrome c are also found inside mitochondria. The level of internalized altered apocytochrome c is decreased in a strain that completely lacks heme lyase and is greatly increased in a strain that overexpresses heme lyase. Antibodies recognizing heme lyase were used to demonstrate that the enzyme is found on the outer surface of the inner mitochondrial membrane and is not enriched at sites of contact between the inner and outer mitochondrial membranes. These results suggest that apocytochrome c is transported across the outer mitochondrial membrane by a freely reversible process, binds to heme lyase in the intermembrane space, and is then trapped inside mitochondria by an irreversible conversion to holocytochrome c accompanied by folding to the native conformation. Altered apocytochrome c lacking the ability to have heme covalently attached accumulates in mitochondria only to the extent that it remains bound to heme lyase.

Apoproteins↗
PubMed1991

Two types of TATA elements for the CYC1 gene of the yeast Saccharomyces cerevisiae.

Functional TATA elements in the 5' untranslated region of the CYC1 gene in the yeast Saccharomyces cerevisiae have been defined by transcriptional analysis of site-directed mutations. Five sites previously suggested to contain functional TATA elements were altered individually and in all possible combinations. The results indicated that only two elements are required for transcription at the normal level and the normal start sites. The two functional TATA elements are located at sites -178 and -123, where the A of the ATG start codon is assigned nucleotide position +1. They direct initiation within windows encompassing -70 to -46 and -46 to -28, respectively. Only when both of the upstream TATA sites were rendered nonfunctional were the third and fourth downstream TATA-like sequences activated, as indicated by the presence of low levels of transcription starting at -28. The two upstream functional TATA elements differed in sequence. The sequence of the most 5' one at site 1, denoted beta-type, was ATATATATAT, whereas that of the second one at site 2, denoted alpha-type, was TATATAAAA. The following rearrangements of the beta-type and alpha-type elements at two sites (1 and 2) were examined: site1 beta-site2 alpha; site 1 alpha-site 2 beta; site1 alpha-site2 alpha; and site1 beta-site2 beta. When different types were at different sites (site1 beta-site2 alpha and site1 alpha-site2 beta), both were used equally. In contrast, when the same type was present at both sites (site1 alpha-site2 alpha and site1 beta-site2 beta), only the upstream element was used. We suggest that the two TATA elements are recognized by different factors of the transcription apparatus.

Base Sequence↗
PubMed1991

An interesting case presentation: ultrasound diagnosis of primary atrial thrombosis associated with brain infarctions.

A premature infant with nonbacterial endocardial thrombosis and embolization of the cerebral circulation is presented. The patient was born at 32 weeks with a birthweight of 1480 g. Pregnancy was complicated by toxemia and placental infarctions. Echocardiographic examination by 12 hours after birth revealed a large mass in the right atrium compatible with endocardial thrombus. Follow-up echocardiograms showed a small mass in the left atrium and a decrease of the right atrial mass by day 7. Echoencephologram studies were normal on day 1. An echodense area in the subcortical white matter was seen by day 2. Other echodensities were detected by day 10. Computerized tomography studies showed areas of decreased attenuation in both hemispheres. Lung perfusion scan and renal-liver ultrasound studies were normal. All these cerebral and cardiac lesions were asymptomatic. Real-time ultrasonography is useful to diagnose asymptomatic endocardial thrombosis and its cerebral complications in premature infants.

Cerebral Infarction↗
PubMed1990

The specificities of yeast methionine aminopeptidase and acetylation of amino-terminal methionine in vivo. Processing of altered iso-1-cytochromes c created by oligonucleotide transformation.

The specificities of methionine aminopeptidase and amino-terminal acetylation in the yeast Saccharomyces cerevisiae were investigated in vivo by sequencing a series of altered iso-1-cytochrome c. Twenty iso-1-cytochromes c, each having a different penultimate residue in the sequence Met-Xaa-Phe-Leu-, were created by transforming yeast directly with synthetic oligonucleotides. The degree of methionine cleavage and amino-terminal acetylation was estimated from the levels of pertinent peptides separated by high performance liquid chromatography. The results confirmed our earlier hypothesis (Sherman, F., Stewart, J. W., and Tsunasawa, S. (1985) BioEssays 3, 27-31) that methionine is completely removed from penultimate residues having radii of gyration of 1.29 A or less (glycine, alanine, serine, cysteine, threonine, proline, and valine). However, only partial cleavage occurred in the sequences Met-Thr-Pro-Leu- and Met-Val-Pro-Leu-, demonstrating that proline at the third position inhibits methionine cleavage when the penultimate residue has an intermediate radius of gyration. Acetylation of the retained amino-terminal methionine occurred completely with the Ac-Met-Glu-Phe-Leu- and Ac-Met-Asp-Phe-Leu- sequences and partially with the Ac-Met-Asn-Phe-Leu-sequence. Although the consensus for acetylation of the retained amino-terminal methionine is not completely known, these results and the results of published sequences indicated that Ac-Met-Glu- and Ac-Met-Asp- (methionine followed by an acidic residue) is sufficient for amino-terminal acetylation in eukaryotes but not in prokaryotes.

Acetylation↗
PubMed1990

Nucleotide sequence of the COR region: a cluster of six genes in the yeast Saccharomyces cerevisiae.

We have determined the nucleotide (nt) sequence of the 7.5-kb COR segment that encompasses a cluster of six genes (CYC1, UTR1, UTR3, OSM1, tRNA(Gly) and RAD7) located on chromosome X of the yeast Saccharomyces cerevisiae. This sequence revealed five open reading frames and a tRNA gene which correspond in position, size and orientation to the transcripts previously identified by Barry et al. [Mol. Cell. Biol. 7 (1987) 632-638]. The extensively studied CYC1 gene encodes iso-1-cytochrome c; the UTR1 and UTR3 genes encode dispensible proteins whose functions are unknown; the OSM1 gene encodes a protein required for growth on hypertonic media; the tRNA(Gly) gene encodes a glycine tRNA; and the RAD7 gene encodes a protein required for repair of UV-induced damage. The OSM1 protein contains a signal sequence for secretion and a region similar to GTP-binding domains. The RAD7 protein displays 5'-untranslated elements similar to those of the stress-inducible gene UB14. The nt sequence upstream from the tRNA(Gly) gene contains a diverged copy of the sigma repeated element. This cluster of COR genes appears to have an ancestral relationship with the cluster of ARC genes on chromosome V.

Amino Acid Sequence↗