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Biomedical subjects

F Sherman

Publications and source records attributed to F Sherman.

At least 127 records · Page 7Linked to original sources
PubMed1988

Effects of amino acid replacements in yeast iso-1 cytochrome c on heme accessibility and intracomplex electron transfer in complexes with cytochrome c peroxidase.

The kinetics of reduction of wild type and several site-specific mutants of yeast iso-1 cytochrome c (Arg-13----Ile, Gln-16----Ser, Gln-16----Lys, Lys-27----Gln, Lys-72----Asp), both free and in 1:1 complexes with yeast cytochrome c peroxidase, by free flavin semiquinones have been studied. Intramolecular one-electron transfer from the ferrous cytochromes c to the H2O2-oxidized peroxidase at both low (8 mM) and high (275 mM) ionic strengths was also studied. The accessibility of the cytochrome c heme within the electrostatically stabilized complex and the rate constants for intramolecular electron transfer at both low and high ionic strength are highly dependent on the specific amino acids present at the protein-protein interface. Importantly, replacement by uncharged amino acids of Arg or Lys residues thought to be important in orientation and/or stabilization of the electron-transfer complex resulted in increased rates of electron transfer. In all cases, an increase in ionic strengths from 8 to 275 mM also produced increased intramolecular electron-transfer rate constants. The results suggest that the electrostatically stabilized 1:1 complex is not optimized for electron transfer and that by neutralization of key positively charged residues, or by an increase in the ionic strength thereby masking the ionic interactions, the two proteins can orient themselves to allow the formation of a more efficient electron-transfer complex.

Amino Acids↗
PubMed1988

Multiple base-pair mutations in yeast.

The nucleotide changes associated with both forward and reverse mutations at the CYC1 locus in the yeast Saccharomyces cerevisiae have been investigated by sequencing the mutated gene product, iso-1-cytochrome c and, more directly, by sequencing appropriate DNA segments. Although the majority of these mutations are the result of single base-pair changes, approximately 10% are the result of multiple mutations and these occur predominantly at certain sites and with certain patterns. Most multiple base-pair changes occur within 20 nucleotides of each other and are generally within six nucleotides. On the basis of the frequencies and patterns of mutations, these nucleotide changes are considered to have occurred as single, concerted events, rather than as multiple independent mutations. Analysis of these mutations indicates that multiple base-pair changes can arise by widely differing mechanisms. We have recognized the following classes of mutations: multiple base-pair changes that yield (1) direct repeats or (2) inverted repeats of local DNA sequences; (3) substitutions of two tandem base-pairs; (4) frameshift and contiguous single base-pair substitutions; and (5) recombination of the CYC1 gene with a non-allelic gene, resulting in alterations within contiguous segments that can be over 150 nucleotides in length. Some of the multiple base-pair changes do not fall into any of these categories. We suggest mechanisms to account for each of these five classes.

Amino Acid Sequence↗
PubMed1988

Yeast iso-1-cytochrome c: genetic analysis of structural requirements.

We describe the use of classical and molecular genetic techniques to investigate the folding, stability, and enzymatic requirements of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae. Interpretation of the defects associated with an extensive series of altered forms of iso-1-cytochrome c was facilitated by the recently resolved three dimensional structure of iso-1-cytochrome c [(1987) J. Mol. Biol. 199, 295-314], and by comparison with the phylogenetic series of eukaryotic cytochromes c. Residue replacements that abolish iso-1-cytochrome c function appear to do so by affecting either heme attachment or protein stability; no replacements that abolish electron transfer function without affecting protein structure were uncovered. Most nonfunctional forms retained at least partial covalent attachment to the heme moiety; heme attachment was abolished only by replacements of Cys19 and Cys22, which are required for thioether linkage, and His23, a heme ligand. Replacements were uncovered that retain function at varying levels, including replacements at evolutionarily conserved positions, some of which were structurally and functionally indistinguishable from wild type iso-1-cytochrome c.

Amino Acid Sequence↗
PubMed1988

Transformation of yeast with synthetic oligonucleotides.

Genomic DNA of the yeast, Saccharomyces cerevisiae, can be conveniently and specifically altered by transforming spheroplasts or lithium acetate-treated cells directly with synthetic oligonucleotides. Altered forms of iso-1-cytochrome c were generated by transforming a cyc1 mutant with oligonucleotides and selecting for at least partially functional revertants; the oligonucleotides contained a sequence that corrected the cyc1 mutation and produced additional alterations at nearby sites. Transformation has been accomplished with oligonucleotides as short as 20 nucleotides and with amounts as low as 100 micrograms. This method of site-directed mutagenesis in vivo has been used to produce alterations in the NH2-terminal region of iso-1-cytochrome c in which the NH2-terminal methionine is excised and the penultimate residue is acetylated.

Alleles↗
PubMed1988

Differential mismatch repair can explain the disproportionalities between physical distances and recombination frequencies of cyc1 mutations in yeast.

Recombination rates have been examined in two-point crosses of various defined cyc1 mutations that cause the loss or nonfunction of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. Recombinants arising by three different means were investigated, including X-ray induced mitotic recombination, spontaneous mitotic recombination, and meiotic recombination. Heteroallelic diploid strains were derived by crossing cyc1 mutants containing a series of alterations at or near the same site to cyc1 mutants containing alterations at various distances. Marked disproportionalities between physical distances and recombination frequencies were observed with certain cyc1 mutations, indicating that certain mismatched bases can significantly affect recombination. The marker effects were more pronounced when the two mutational sites of the heteroalleles were within about 20 base pairs, but separated by at least 4 base pairs. Two alleles, cyc1-163 and cyc1-166, which arose by G.C----C.G transversions at nucleotide positions 3 and 194, respectively, gave rise to especially high rates of recombination. Other mutations having different substitutions at the same nucleotide positions were not associated with abnormally high recombination frequencies. We suggest that these marker effects are due to the lack of repair of either G/G or C/C mismatched base pairs, while the other mismatched base pair of the heteroallele undergoes substantial repair. Furthermore, we suggest that diminished recombination frequencies are due to the concomitant repair of both mismatches within the same DNA tract.

Alleles↗
PubMed1988

A highly revertible cyc1 mutant of yeast contains a small tandem duplication.

A mutant, cyc1-96, that reverts spontaneously at an extremely high rate, was uncovered after examining approximately 500 cyc1 mutants which lack or have defective iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. Cloning and DNA sequencing of appropriate fragments revealed that the cyc1-96 mutation contained a 19 bp duplication whereas the spontaneously arising revertants contained the normal wild-type sequence. Because the 19 bp segment in the wild-type sequence is flanked by a 5 bp repeat and because the cyc1-96 mutation arose spontaneously, the 19 bp duplication may have arisen by slippage and misalignment during DNA synthesis. The high reversion rate was not diminished in strains containing the rad52 mutation, which generally reduces mitotic recombination, including recombination associated with the elimination of a segment of a long direct repeat. Thus the loss of segments from short and long duplications occur by different mechanisms. We suggest that the high reversion rates of cyc1-96 and other short duplications are due to misalignment errors during replication.

Amino Acid Sequence↗
PubMed1988

Efficiency of translation initiation by non-AUG codons in Saccharomyces cerevisiae.

The quantitative levels of initiation of protein synthesis at codons other than AUG were determined with a CYC7-lacZ fused gene in the yeast Saccharomyces cerevisiae. AUG was the only codon which efficiently initiated translation, although some non-AUG codons allowed initiation at very low efficiency, below 1% of the normal level. Since translation initiates at codons other than AUG in at least two wild-type genes from eucaryotes, other factors presumably play a role in enhancing the activity of non-AUG codons.

Base Sequence↗
PubMed1988

mRNA structures influencing translation in the yeast Saccharomyces cerevisiae.

The mRNA sequence and structures that modify and are required for translation of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae were investigated with sets of CYC1 alleles having alterations in the 5' leader region. Measurements of levels of CYC1 mRNA and iso-1-cytochrome c in strains having single copies of altered alleles with nested deletions led to the conclusion that there is no specific sequence adjacent to the AUG initiator codon required for efficient translation. However, the nucleotides preceding the AUG initiator codon at positions -1 and -3 slightly modified the efficiency of translation to an order of preference similar to that found in higher cells. In contrast to large effects observed in higher eucaryotes, the magnitude of this AUG context effect in S. cerevisiae was only two- to threefold. Furthermore, introduction of hairpin structures in the vicinity of the AUG initiator codon inhibited translation, with the degree of inhibition related to the stability and proximity of the hairpin. These results with S. cerevisiae and published findings on other organisms suggest that translation in S. cerevisiae is more sensitive to secondary structures than is translation in higher eucaryotes.

Alleles↗
PubMed1987

Replacement of the invariant lysine 77 by arginine in yeast iso-1-cytochrome c results in enhanced and normal activities in vitro and in vivo.

Oligonucleotide-directed mutagenesis of the yeast Saccharomyces cerevisiae was used to generate an abnormal iso-1-cytochrome c having an Arg-77 replacement of the normal Lys-77; this Lys-77 residue is evolutionarily conserved in most eukaryotic cytochromes c and is trimethylated in fungal and plant cytochromes c. Examination of strains having a single chromosomal copy of the gene encoding the Arg-77 protein indicated that the altered protein was synthesized at the normal rate and that it had normal or near normal activity in vivo. Examination of enzymatic activities in vitro with cytochrome b2, cytochrome c peroxidase, and cytochrome c oxidase indicated that the altered iso-1-cytochrome c has equal or enhanced catalytic efficiencies. Thus, replacement of the evolutionarily conserved residue Lys-77 produces no or only minor effects both in vivo and in vitro.

Amino Acid Sequence↗
PubMed1987

Identification and sequence of the gene encoding cytochrome c heme lyase in the yeast Saccharomyces cerevisiae.

Mitochondrial cytochrome c contains a heme group covalently attached through thioether linkages to two cysteinyl residues of the protein. We demonstrate here that the nuclear gene, CYC3, in the yeast Saccharomyces cerevisiae, encodes cytochrome c heme lyase (CCHL), the enzyme catalyzing the attachment of heme to apocytochrome c. Mitochondrial extracts from cyc3- mutants are deficient in CCHL activity compared with extracts from normal strains, whereas strains carrying multiple copies of the CYC3 gene exhibit high levels of the activity. The CYC3 gene was cloned by functional complementation of a cyc3- mutant using a previously isolated plasmid containing the gene PYK1, which is tightly linked to CYC3. An open reading frame encoding a protein of 269 amino acids was identified from the DNA sequence of a fragment encompassing the CYC3 gene, and the corresponding transcript shown to be approximately 0.9 kb in length. CCHL appears to be a single polypeptide chain which acts specifically on the two forms of cytochrome c, but not on cytochrome c1.

Amino Acid Sequence↗
PubMed1987

Accuracy of subcostal two-dimensional echocardiography in prospective diagnosis of total anomalous pulmonary venous connection.

Since reparative surgery without cardiac catheterization has been advocated for certain lesions such as total anomalous pulmonary venous connection (TAPVC), it is important to assess the accuracy of two-dimensional (2-D) echocardiography in the prospective diagnosis of this lesion. Abandoning traditional cardiac catheterization and angiography would be justified only if all surgically relevant anatomic features could be consistently displayed noninvasively. Consequently, we reviewed our experience with TAPVC to determine the suitability of subcostal 2-D echocardiography as the sole prospective diagnostic tool. Over a 5-year period, of 2444 infants under 2 years of age who underwent subcostal 2-D echocardiography, 38 were diagnosed as having TAPVC as the only major cardiac defect. There were no false-negative or false-positive cases during the time of the study. Drainage sites were diagnosed correctly in 36 (95%). Of the five cases of mixed-type TAPVC, the second drainage site was missed by 2-D echocardiography in two cases.

Echocardiography↗
PubMed1987

Physical analysis of the COR region: a cluster of six genes in Saccharomyces cerevisiae.

Six genes, CYC1, UTR1, UTR3, OSM1, tRNAGly, and RAD7, have been localized within an 8-kilobase region on chromosome X of the yeast Saccharomyces cerevisiae. The physical structures and the transcripts of these genes were identified by analyzing a normal strain and six deletion mutants by genomic blotting, transcriptional analysis, and gene disruption procedures. The well-studied CYC1 gene encodes iso-1-cytochrome c; the tRNAGly gene encodes a tRNA; deletion of OSM1 and RAD7 causes sensitivity to hypertonic medium and UV irradiation, respectively. There were no observable phenotypes in strains having deletions of the UTR1, UTR3, and tRNAGly gene. The high density of transcripts, with little or almost no intragenic regions, indicates that the chromosomal organization of S. cerevisiae resembles the chromosomal organization of procaryotes rather than higher eucaryotes.

Chromosome Mapping↗
PubMed1986

Guanidine hydrochloride induced equilibrium unfolding of mutant forms of iso-1-cytochrome c with replacement of proline-71.

Proline-71, an evolutionally conserved residue that separates two short alpha-helical regions, is replaced by valine, threonine, or isoleucine in at least partially functional forms of iso-1-cytochrome c from Saccharomyces cerevisiae [Ernst, J. F., Hampsey, D. M., Stewart, J. W., Rackovsky, S., Goldstein, D., & Sherman, F. (1985) J. Biol. Chem. 260, 13225-13236]. Treatment of these proteins with a specific sulfhydryl blocking reagent (methyl methanethiosulfonate) to block Cys-102 has allowed investigation of the properties of monomeric forms of the proteins, denoted iso-1-MS. Comparison of the UV-visible absorbance properties (pH 6, 20 degrees C) shows minor differences between the normal Pro-71 iso-1-MS and two of the three mutant proteins. The Val-71 iso-1-MS protein has absorbance properties indistinguishable from those of the normal Pro-71 iso-1-MS protein, but the Ile-71 iso-1-MS and Thr-71 iso-1-MS proteins show reduced intensity of the 695-nm absorbance band and a small shift in the Soret maximum, from 408 nm for the Pro-71 iso-1-MS and Val-71 iso-1-MS proteins to 406 nm for the Thr-71 iso-1-MS and Ile-71 iso-1-MS proteins. Second derivative spectroscopy is used to assess differences in the polarity of the environment of tyrosine residues. The average degree of exposure of tyrosines to solvent is similar in all four proteins: 0.39 for the normal Pro-71 iso-1-MS and Val-71 iso-1-MS proteins; 0.40 for the Ile-71 iso-1-MS protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome c Group↗
PubMed1986

Altered 40 S ribosomal subunits in omnipotent suppressors of yeast.

The five suppressors SUP35, SUP43, SUP44, SUP45 and SUP46, each mapping at a different chromosomal locus in the yeast Saccharomyces cerevisiae, suppress a wide range of mutations, including representatives of all three types of nonsense mutations, UAA, UAG and UGA. We have demonstrated that ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46 translate polyuridylate templates in vitro with higher errors than ribosomes from the normal stain, and that this misreading is substantially enhanced by the antibiotic paromomycin. Furthermore, ribosomal subunit mixing experiments established that the 40 S ribosomal subunit, and this subunit only, is responsible for the higher levels of misreading. Thus, the gene products of SUP35, SUP44, SUP45 and SUP46 are components of the 40 S subunit or are enzymes that modify the subunit. In addition, a protein from the 40 S subunit of the SUP35 suppressor has an altered electrophoretic mobility; this protein is distinct from the altered protein previously uncovered in the 40 S subunit of the SUP46 suppressor. In contrast to the ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46, the ribosomes from the SUP43 suppressor do not significantly misread polyuridylate templates in vitro, suggesting that this locus may not encode a ribosomal component or that the misreading is highly specific.

Centrifugation, Density Gradient↗
PubMed1986

Amino acid replacements in yeast iso-1-cytochrome c. Comparison with the phylogenetic series and the tertiary structure of related cytochromes c.

The structural and folding requirements of eukaryotic cytochromes c have been investigated by determining the appropriate DNA sequences of a collection of 46 independent cyc 1 missense mutations obtained in the yeast Saccharomyces cerevisiae and by deducing the corresponding amino acid replacements that abolish function of iso-1-cytochrome c. A total of 33 different replacements at 19 amino acid positions were uncovered in this and previous studies. Because all of these nonfunctional iso-1-cytochromes c are produced at far below the normal level and because a representative number are labile in vitro, most of the replacements appear to be affecting stability of the protein or heme attachment. By considering the tertiary structure of related cytochromes c, the loss of function of most of the mutant iso-1-cytochromes c could be attributed to either replacements of critical residues that directly interact with the heme group or to replacements that disrupt the proper folding of the protein. The replacements of residues interacting with the heme group include those required for covalent attachment (Cys-19 and Cys-22), ligand formation (His-23 and Met-85), and formation of the immediate heme environment (Leu-37, Tyr-53, Trp-64, and Leu-73). Proper folding of the protein is prevented by replacements of glycine residues at sites that cannot accommodate side chains (Gly-11 and Gly-34); by replacements of residues with proline, which limit the torsion angle (Leu-14 and His-38); and by replacements apparently unable to direct the local folding of the backbone into the proper conformation (Pro-35, Tyr-72, Asn-75, Pro-76, Lys-84, Leu-99, and Leu-103). Even though most of the missense mutations occurred at sites corresponding to evolutionarily invariant or conserved residues, a consideration of the replacements in functional revertants indicates that the requirement for residues evolutionarily preserved is less stringent than commonly assumed.

Alleles↗
PubMed1986

Mutational alterations induced in yeast by ionizing radiation.

The cycl-9 ochre (UAA) mutant and the cycl-179 amber (UAG) mutant of the yeast Saccharomyces cerevisiae were reverted with X-rays and alpha-particles. The amino acid sequence changes of iso-1-cytochromes c from 36 of the intragenic revertants were determined by amino acid analysis and peptide mapping, aided by partial amino acid sequencing of 4 revertants. In addition, the DNA segments encompassing 3 unusual mutations with complex changes were cloned and sequenced. This study and previous studies of 16 other revertants of cycl-9 and cycl-179 revealed that ionizing radiation primarily induces single base-pair substitutions; 47 of the 52 revertants arose by transversions and transitions without any apparent preference. However, the A X T----T X A substitution at the first base pair for the cycl-179 UAG codon, leading to the normal protein, was not detected, nor was it found previously in 32 revertants of cycl-179 obtained spontaneously or induced with various other mutagens; apparently, there is a prohibition of certain base-pair substitutions at certain sites in DNA. In addition, 5 of the 52 revertants arose by multiple changes within a short region of 11 base pairs. These consisted of the deletion of 6 base pairs, the substitution of 3 base pairs, and 3 different kinds of substitutions of two base pairs. Compared to other mutagens previously tested with the cycl system, ionizing radiation produces the most random types of base-pair substitutions.

Alpha Particles↗