Nuclear pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe strictly requires an intron-contained, conserved sequence element.

It has recently been argued that pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe may be more similar to splicing in metazoan species than in the budding yeast Saccharomyces cerevisiae. In this report we show that, contrary to this assumption, the conserved sequence element 5'-CTPu APy-3' found in all S. pombe introns 6-18 nucleotides upstream of the 3' splice site is, like the TACTAAC box in S. cerevisiae, indispensable for efficient splicing. The conserved adenine residue of this sequence is used for branch formation and point mutations introduced into the CTPuAPy sequence abolish splicing and seem not to result in the recruitment of cryptic branch sites. We also show that an S. cerevisiae intron is correctly excised in S. pombe whereby the TACTAAC box is used in branch formation.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Characterization of an amidated form of pancreatic polypeptide from the daddy sculpin (Cottus scorpius)

The primary structure of pancreatic polypeptide from the teleostean fish, Cottus scorpius (daddy sculpin) was established as: YPPQPESPGGNASPEDWAKYHAAVRHYVNLITRQRYNH2 The presence of a COOH-terminally alpha-amidated amino acid was established using an HPLC method of general applicability. Although the peptide shows strong homology towards anglerfish pancreatic polypeptide (86%), homology towards porcine peptide YY (PYY) (61%) and porcine neuropeptide Y (NPY) (61%) was greater than towards porcine pancreatic polypeptide (PP) (47%). This result supports suggestions that the gene duplication events which led to PP, NPY and PYY formation took place after the time of divergence of fish and mammals.     

Structure of two human beta-actin-related processed genes one of which is located next to a simple repetitive sequence.

From a human gene library we have isolated and sequenced a beta-actin-like pseudogene, H beta Ac-psi 2, which lacks intervening sequences and contains several mutations resulting in frame-shifts, stop codons and in a departure from the known beta-actin protein sequence. We have also extended our sequence work on the intronless human beta-actin-related pseudogene H beta Ac-psi 1 described previously and we find that both genes are processed genes ending in a poly(dA) tract and flanked by direct repeats. The gene H beta Ac-psi 2 is preceded by a 230-bp region in which the simple sequence 5'-GAAA-3' is repeated greater than 40 times. This satellite-like sequence is highly repetitive in the human genome.     

Effects of cordycepin,hydroxyurea and cycloheximide on histone mRNA synthesis in synchronized HeLa cells

The effects of cordycepin, hydroxyurea and cycloheximide on the synthesis of histone mRNA in synchronized HeLa cells were studied by quantitating the RNA, using translation in a rabbit reticulocyte cell-free system. It was found that biologically active histone mRNA is synthesized in the presence of cordycepin. This result strengthens the findings by others that histone mRNA does not contain poly(A). Hydroxyurea and cycloheximide, when used at concentrations that inhibit cellular DNA synthesis, had different effects on histone messenger activity. While polyribosomal messenger activity rapidly declined after addition of hydroxyurea it was not impaired by cycloheximide. These results might help to elucidate regulatory mechanisms involved in the coupled synthesis of DNA and histones in HeLa cells.     

Structural and functional analysis of ypt2, an essential ras-related gene in the fission yeast Schizosaccharomyces pombe encoding a Sec4 protein homologue.

Using the cloned Saccharomyces cerevisiae YPT1 gene as hybridization probe, a gene, designated ypt2, was isolated from the fission yeast Schizosaccharomyces pombe and found to encode a 200 amino acid long protein most closely related to the ypt branch of the ras superfamily. Disruption of the ypt2 gene is lethal. The bacterially produced ypt2 gene product is shown to bind GTP. A region of the ypt2 protein corresponding to but different from the 'effector region' of ras proteins is also different from that of ypt1 proteins of different species but identical to the 'effector loop' of the S.cerevisiae SEC4 gene product, a protein known to be required for vesicular protein transport. The S.pombe ypt2 gene under control of the S.cerevisiae GAL10 promoter is able to suppress the temperature-sensitive phenotype of a S. cerevisiae sec4 mutant, indicating a functional similarity of these GTP-binding proteins from the two very distantly related yeasts.     

Identification and partial purification of GTPase-activating proteins from yeast and mammalian cells that preferentially act on Ypt1/Rab1 proteins

Two GTPase-activating proteins of apparent molecular mass of 100 kDa and 30 kDa have been partially purified from porcine liver cytosol using mammalian Ypt1/Rab1 protein as substrate. Both proteins act most efficiently on Ypt1/Rab1p, but are inactive with H-Ras p21. From the budding yeast Saccharomyces cerevisiae, a cytosolic 40 kDa yptGAP was partially purified. It accelerates the intrinsic GTPase activity of wild-type Ypt1p but not of H-Ras p21 or a mutant ypt1p with an amino acid substitution of the effector domain which renders the protein functionally inactive in yeast cells.     

Preparation of an isomorphous heavy-atom derivative of tobacco mosaic virus by chemical modification with 4-sulpho-phenylisothiocyanate

The reaction of the vulgare and U2 strains of tobacco mosaic virus with 4-sulpho-phenylisothiocyanate has been investigated. The coat protein of the U2 strain has a proline residue at its N-terminus and a lysine residue at position 53. Whereas both residues could be reacted with 4-sulpho-phenylisothiocyanate in the isolated coat protein, only proline-1 was modified during treatment of the intact virus with the same reagent, thereby showing that the loss of reactivity of the ?-amino group of lysine-53 is a consequence of the virus structure. The 4-sulpho-phenylthiocarbamoyl derivative of amino groups shows considerable tautomerism and, as a consequence, it proved possible to prepare a heavy-atom derivative of the intact U2 strain in which methyl mercury nitrate was bound by the modified N-terminal residue of the coat protein.On the other hand, when the intact vulgare strain was treated with 4-sulphophenylisothiocyanate, little or no modification of the ?-amino groups of the two lysine residues (positions 53 and 68) per polypeptide chain was observed. Taking into account previous studies on the reactivity of the amino groups of the coat protein in tobacco mosaic virus vulgare and assuming that all strains and mutants have closely similar three-dimensional structures, these experiments suggest that the N-terminal residue is more exposed (i.e. probably nearer the virus “surface”) than the side-chain of lysine-68, which in turn is more accessible than the side-chain of lysine-53. This interpretation is readily compatible with the results of X-ray diffraction analysis carried out on these chemically modified viruses (Mandelkow &; Holmes, 1974) and lends support to the hope that such methods of preparing heavy-atom derivatives of proteins will be of general use.