Identification of the predominant non-native histidine ligand in unfolded cytochrome c.

The heme and its two axial ligands, His18 and Met80, play a central role in the folding/unfolding mechanism of cytochrome c. Because of the covalent heme attachment, His18 remains bound under typical denaturing conditions, while the more labile Met80 ligand is replaced by an alternate histidine ligand. To distinguish between the two possible non-native histidine ligands in horse cytochrome c, variants with a His26 to Gln or His33 to Asn substitution were prepared using a yeast expression system.

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 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.

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.

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.

Chromosomal assignment of mutations by specific chromosome loss in the yeast Saccharomyces cerevisiae.

Yeast 2-microns plasmids were integrated near the centromere of a different chromosome in each of 16 cir0 mapping strains of Saccharomyces cerevisiae. The specific chromosomes containing the integrated 2-microns plasmid DNA were lost at a high frequency after crossing the cir0 strains to cir+ strains. A recessive mutation in a cir+ strain can then be easily assigned to its chromosome using this set of mapping strains, since the phenotype of the recessive mutation will be manifested only in diploids having the integrated 2-microns plasmid and the unmapped mutation on homologous chromosomes.

Isolation and characterization of omnipotent suppressors in the yeast Saccharomyces cerevisiae.

Approximately 290 omnipotent suppressors, which enhance translational misreading, were isolated in strains of the yeast Saccharomyces cerevisiae containing the psi+ extrachromosomal determinant. The suppressors could be assigned to 8 classes by their pattern of suppression of five nutritional markers. The suppressors were further distinguished by differences in growth on paromomycin medium, hypertonic medium, low temperatures (10 degrees), nonfermentable carbon sources, alpha-aminoadipic acid medium, and by their dominance and recessiveness.

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.

A new suppressor of mutations in the DNA repair-recombination genes of bacteriophage T4: sur.

A new mutation designated sur was isolated as a suppressor of a mutation in the uvsX gene of T4 phage. Unlike the other suppressors of mutations in genes involved in DNA repair and recombination, sur has a wide range, suppressing both DNA repair and replication defects in mutations in genes uvsX, uvsY, 46, 47, and 59. However, its suppressor functions may be confined to the uvsX-uvsY DNA repair pathway since sur did not suppress a mutation in the denV gene.

An analysis of DNA repair and recombination functions of bacteriophage T4 by means of suppressors: the role of das.

Previous studies have indicated that the bacteriophage T4 das mutations partially suppressed the DNA replication defects in gene 46 and 47 mutations. Here it is shown that the das mutation also suppresses the DNA repair defects but not the DNA replication defects of the uvsX and uvsY mutations. In contrast, the das mutation suppressed both the DNA replication and repair defects of gene 46 and 47 mutations.

The coupling of DNA repair-recombination functions with DNA replication in bacteriophage T4: a new DNA repair mutant.

The requirement of DNA repair-recombination functions for T4 phage DNA replication has been known for some time but the underlying basis for this relationship has been unclear. This report is concerned with a new uv-sensitive gene [uvsU], whose function appears to bridge these two major activities of DNA. The [uvsU] mutant fails to complement [uvsX] mutants but uvsU maps in a region distinct from uvsX.