FAS1, the structural gene of the pentafunctional fatty acid synthetase subunit beta in Saccharomyces cerevisiae has been sequenced. Its reading frame represents an intron-free nucleotide sequence of 5,535 base pairs, corresponding to a protein of 1,845 amino acids with a molecular weight of 205,130 daltons. In addition to the coding sequence, 1,468 base pairs of its 5'-flanking region were determined. S1 nuclease mapping revealed two transcriptional initiation sites; 5 and 36 base pairs upstream of the translational start codon. Within the flanking sequences two TATATAAA boxes, several A-rich and T-rich blocks and a TAG...TATGTT...TATGTT...TTT sequence were found and are discussed as transcriptional initiation and termination signals, respectively. The order of catalytic domains in the cluster gene was established by complementation of defined fas1 mutants with overlapping FAS1 subclones. Acetyl transferase (amino acids 1-468) is located proximal to the N-terminus of subunit beta, followed by the enoyl reductase (amino acids 480-858), the dehydratase (amino acids 1,134-1,615) and the malonyl/palmityl transferase (amino acids 1,616-1,845) domains. One major inter-domain region of about 276 amino acids with so far unknown function was found between the enoyl reductase and dehydratase domains. The substrate-binding serine residues of acetyl, malonyl and palmityl transferases were identified within the corresponding domains. Significant sequence homologies exist between the acyl transferase active sites of yeast and animal fatty acid synthetases. Similarly, a putative sequence of the enoyl reductase active site was identified.
From a Saccharomyces cerevisiae gene bank contained in the novel yeast cosmid shuttle vector pMS201 the fatty acid synthetase (FAS) genes FAS1 and FAS2 were isolated. FAS clones were identified by in situ colony hybridization using two yeast DNA probes apparently capable of producing avian FAS cross-reacting material (J. Carbon, personal communication). Classification as FAS1 or FAS2 clones was achieved by their specific transformation of fas1 and fas2 yeast mutants. By transcription mapping FAS1 was assigned to about 5.3 kb within 14.8 kb of chromosomal DNA covered by two genomically adjacent BamHI fragments. The FAS2 gene was localized on a single BamHI fragment of 25 kb. One of the FAS clones ( FAS2 ) produces immunologically cross-reacting material in Escherichia coli. High frequency transformation of fas1 mutants was only observed with one subclone, pMS3021 , containing the intact FAS1 locus. Other DNA segments cloned in the same self-replicating vector but representing only part of FAS1 exhibited drastically lower transformation rates. As evident from this and from FAS1 /TRP1-cotransformation rates only the intact FAS1 gene in pMS3021 is capable of fas1 -mutant complementation. With partial FAS1 genes, even when coding for an intact equivalent of the mutated domain, their chromosomal integration is necessary for the expression of FAS. In integrative transformants the coexistence of integrated and autonomously replicating plasmid DNA was demonstrated. Both, the extrachromosomal and chromosomally integrated FAS DNA was mitotically unstable. Transformation studies using subcloned FAS1 DNA segments revealed the relative locations of the enoyl reductase and dehydratase domains within this pentafunctional cluster gene.
The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase)have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties.
Rat α1‐inhibitor 3 clones were isolated by immunological screening of a λ gt11 cDNA library prepared from rat liver poly(A)‐rich RNA. The recombinant cDNA clones were identified by the absence of their immunoprecipitable products following hybrid‐arrested in vitro translation. The size of the cognate poly(A)‐rich RNA was estimated to be roughly 5000 residues. Approximately 16 h after induction of inflammation the amount of α1‐inhibitor 3 poly(A)‐rich RNA decreases as shown by dot‐blot hybridization and Northern analyses. The response of this negative acute‐phase plasma protein to inflammation may therefore be considered to be at the pretranslational level.
The characterized DNA constitutes an open reading frame of 225 amino acids followed by a canonical eucaryotic polyadenylation signal and a poly(A) tail. Sequence microheterogeneity, particularly in the 3′‐flanking region was observed. An amino acid homology of 70% for α1‐inhibitor 3 with human and rodent α2‐macroglobulin emphasizes the evolutionary relationship of the macroglobulins.
A mutation in Escherichia coli K12 giving resistance to about 5/tg/ml of streptomycin was found to be cotransducible by P1 with pro A and proB, and is located at about 8-5 min on the chromosome map. The locus is named strB. A second mutation to the same resistance level was not cotransducible with either pro A or proB and must be located elsewhere. Both mutations cause a marked increase in R-factor mediated streptomycin resistance, and significant decreases in resistance to several other antibiotics, both in the presence and absence of an R-factor determinant for the same antibiotic. The two mutations differ in their effects on bacterial sensitivity to crystal violet and EDTA.
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