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The let-60 ras gene of Caenorhabditis elegans is required for multiple aspects of development. The vulvar differentiation pathway is the most intensively studied of these, but the ras pathway has now been shown to also be essential for male spicule development. Using vulval differentiation, molecular genetic techniques are now being used to study structure/function relationships of particular signaling components and to identify new positively and negatively acting proteins of Ras-mediated signaling pathways. Mutations affecting LET-23, a receptor tyrosine kinase homolog, which cause tissue-specific defects have been localized to the carboxyl terminus. SH2 domain specificity has been analyzed through Src/SEM-5 chimeric proteins in transgenic nematodes. A mitogen-activated protein kinase that acts downstream of LET-60 Ras in vulval differentiation has been identified. Negative regulatory genes have been cloned and found to encode novel proteins and a clathrin adaptor protein.

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To search for histone domains that may regulate transcription in vivo, we made deletions and amino acid substitutions in the histone N-termini of S. cerevisiae. Histone H4 N-terminal residues 4-23, which include the extremely conserved, reversibly acetylated lysines (at positions 5, 8, 12, and 16), were found to encompass a region required for the activation of the GAL1 promoter. Deletions in the H4 N-terminus reduce GAL1 activation 20-fold. This effect is specific to histone H4 in that large deletions in the N-termini of H2A, H2B, and H3 do not similarly decrease induction. Activation of the PHO5 promoter is reduced approximately 4- to 5-fold by these H4 deletions. Mutations in histone H4 acetylation sites and surrounding residues can cause comparable and, in some cases, even greater effects on induction of these two promoters. We postulate that the H4 N-terminus may interact with a component of the transcription initiation complex, allowing nucleosome unfolding and subsequent initiation.

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Repression of transcription from the silent mating loci (HML alpha and HMRa) is essential for mating ability in Saccharomyces cerevisiae. This silencing is known to require at least five proteins (SIR1, SIR2, SIR3, SIR4, and histone H4) and is accompanied by a change in chromatin structure. We show here that four positions of histone H4 (N-terminal residues 16, 17, 18, and 19) are crucial to silencing. HML alpha and HMRa are efficiently repressed when these positions are occupied by basic amino acids but are derepressed when substituted with glycine. These results suggest that acetylation of Lys-16 would lead to derepression of the silent mating loci. Three strong extragenic suppressors of the latter H4 mutations were isolated and determined to be located in SIR3. These suppressors allow high mating efficiencies in cells expressing either wild-type H4 or H4 containing single amino acid substitutions. They did not allow efficient mating in a strain that contained an H4 N-terminal deletion. These results indicate that the SIR3 mutations do not bypass the requirement for the H4 N terminus but, rather, allow repression in the presence of a less than optimal H4 N terminus. This provides a link between one of the SIR proteins and a component of chromatin.

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A gene from Saccharomyces cerevisiae has been mapped, cloned, sequenced and shown to encode a catalytic subunit of an N-terminal acetyltransferase. Regions of this gene, NAT1, and the chloramphenicol acetyltransferase genes of bacteria have limited but significant homology. A nat1 null mutant is viable but exhibits a variety of phenotypes, including reduced acetyltransferase activity, derepression of a silent mating type locus (HML) and failure to enter G0. All these phenotypes are identical to those of a previously characterized mutant, ard1. NAT1 and ARD1 are distinct genes that encode proteins with no obvious similarity. Concomitant overexpression of both NAT1 and ARD1 in yeast causes a 20-fold increase in acetyltransferase activity in vitro, whereas overexpression of either NAT1 or ARD1 alone does not raise activity over basal levels. A functional iso-1-cytochrome c protein, which is N-terminally acetylated in a NAT1 strain, is not acetylated in an isogenic nat1 mutant. At least 20 other yeast proteins, including histone H2B, are not N-terminally acetylated in either nat1 or ard1 mutants. These results suggest that NAT1 and ARD1 proteins function together to catalyze the N-terminal acetylation of a subset of yeast proteins.

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Yeast histone H4 function was probed in vivo by deleting segments of this extremely conserved 102 amino acid protein. Deletions in the hydrophobic core of H4 are lethal and block chromosomal segregation. In contrast, deletions at the hydrophilic N terminus (residues 4-28) and C terminus (residues 100-102) are viable. However, N-terminal deletion alters normal chromatin structure and lengthens the cell cycle, especially G2. Surprisingly, removal of the H4 N terminus also derepresses the silent mating type loci, HML alpha and HMRa, disrupting mating. This activation is specific since other regulated genes (GAL10, PHO5, CUP1) are repressed and induced normally in these cells. Deletions of the hydrophilic N termini of H2A or H2B do not show this effect on mating. These experiments allow us to define a unique H4 function that is not shared by other histones (H2A and H2B).

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To aid in the interpretation of the 3.5 A resolution electron density map of glutamine synthetase (GS) from Salmonella typhimurium, the nucleotide sequence of the gene coding for this enzyme has been determined. The predicted sequence of 468 amino acids (Mr = 51,628) has been compared to the sequence and sequence fragments reported by others for GS of Anabaena and Escherichia coli. The homology between the pairs of sequences is sufficiently strong to suggest that the overall three-dimensional structures of the three GS are similar. The predicted positions of alpha helices are in moderately good agreement with the electron-density map.

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Four fragments from the maxicircle DNA of Leishmania tarentolae cloned into the selectable Saccharomyces cerevisiae shuttle vector, YIp5, exhibited autonomous replicating sequence (ars) activity. Two of the fragments (pSK120, pSK152) produced large yeast transformant colonies and two (pSK30, pSK150) produced small colonies. All yeast transformants contained extrachromosomal self replicating YIp5 hybrid plasmids as shown by mitotic instability in non selective medium and by the transformation of Escherichia coli with yeast minilysates and recovery of the plasmid from the transformed bacteria. The copy numbers of pSK30, pSK150 and pSK152 in the transformed yeast were approximately the same as that of the YRp12 control, which contains the yeast arsl element; the copy number of pSK120, however, was at least 10 fold lower. A 1.87 kb subfragment of the pSK120 fragment also showed strong ars activity. The entire DNA sequences of the pSK120, pSK152 and pSK150 fragments are known, and several yeast 11 mer consensus ars sequences are present within each fragment. In addition there is a sequence (Lt ars 189) within the pSK152 subclone that has 78% similarity with a 189 nt sequence of an ars element from the Crithidia fasciculata maxicircle (Cf ars 189), implying an evolutionary conservation of this putative origin of replication in at least two different kinetoplastid species. The relative positions of the Lt ars 189 sequence in the L. tarentolae maxicircle map and the Cf ars 189 sequence in the C. fasciculata map with respect to the 9 and 12 S ribosomal genes are similar, implying an overall conservation of gene order in this portion of the transcribed regions of these two species and perhaps in all kinetoplastid species.

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