We identified a P element insertional mutant of the Drosophila glycogen phosphorylase (DGPH) gene. Glycogen phosphorylase protein concentration and enzyme activity are decreased while glycogen ...content is increased in flies homozygous for the mutant allele. The DGPH gene has been cloned and sequenced; its open reading frame codes for a protein of 844 amino acids with a predicted molecular mass of 97 kDa. Comparison of the conceptual amino acid sequence of the Drosophila glycogen phosphorylase with glycogen phosphorylase sequences from other organisms shows a high degree of homology to mammalian enzymes. All the residues of the allosteric effector binding sites, the active site, and the site of phosphorylation are exactly conserved, but some of the residues of the glycogen storage site are not.
A 94 kDa nuclear-localization-signal (NLS)-binding protein was purified from Drosophila embryos. The NLS of the simian-virus-40 T-antigen is specifically bound by the dephosphorylated form of the ...protein. After phosphorylation, the affinity of the protein for the NLS is sharply decreased. In the dephosphorylated form, p94 (protein of 94 kDa) is the major NLS-binding protein in Drosophila embryos. Immunoprecipitation confirmed the ATP-dependent phosphorylation of p94, and co-precipitation of two additional phosphorylated proteins, indicated that the NLS-binding protein is part of a larger complex in Drosophila embryos. In agreement with the immunoprecipitation results, cross-linking experiments demonstrated the interaction of p94 with three additional proteins. These protein-protein interactions were also phosphorylation-dependent.
We isolated and characterized the first chromosomespecific satellite DNA (HC2sat) of Chinese hamster. This novel satellite was localized to the pericentric region of hamster chromosome 2. The 2.8 kb ...long repeat unit of HC2sat was identified and two such units were sequenced. Extended shortrange periodicity could not be revealed in repeat units. These elements are amongst the largest satellite repeat units reported from mammals to date. HC2sat is a major constituent of the pericentric region of CHO chromosome 2 representing a 7 – 14 Mb long DNA segment. Studies of long range organization of HC2sat indicated that the formation of the satellite array might occur in different phases and involved different amplification mechanisms.
Pig muscle 3‐phosphoglycerate kinase contains seven cysteine residues/molecule enzyme. Two of them react with Ellman's reagent (Nbs2) in a second‐order reaction k= (1.1 ± 0.1) × 103 M−1 s−1; the ...reaction of the other five thiols are limited by a first‐order protein structural change k= (2.0 ± 0.4) × 10−4 s−1 in 0.1 M Tris/HCl buffer, pH 7.5 at 20°C.
Blocking the rapidly reacting thiols with Nbs2 inactivated the enzyme (these two –SH groups are not equivalent in this respect), but it does not abolish substrate‐binding ability.
The rapidly reacting thiol groups readily participate in intermolecular disulfide formation following their partial blocking with Nbs2. This type of aggregation of 3‐phosphoglycerate kinase molecules also leads to inactivation.
The order of effectivity of substrates in inhibiting the reaction of the slowly reacting thiols is very similar to the order of their protective effect against heat inactivation. Both phenomena presumably reflect the structure‐stabilizing effect of substrates.
A 1761 base pairs long artificial gene coding for human serum albumin (HSA) has been prepared by a newly developed synthetic approach, resulting in the largest synthetic gene so far described. ...Oligonucleotides corresponding to only one strand of the HSA gene were prepared by chemical synthesis, while the complementary strand was obtained by a combination of enzymatic and cloning steps. 24 synthetic, 69-85 nucleotides long oligonucleotides covering the major part of the HSA gene (41-1761 nucleotides) were used as building blocks. Generally, four groups of 6-6 such oligonucleotides were successively cloned in pUC19 Escherichia coli vector to obtain about quarters of the gene as large fragments. Joining of these four fragments resulted in a cloned DNA coding for the 13-585 amino acid region of HSA, which was further supplemented with a double-stranded linker sequence coding for the amino terminal 12 amino acids. The completed structural gene composed of frequently used codons in the highly expressed yeast genes was then supplied with yeast regulatory sequences and the HSA expression cassette so obtained was inserted into an Escherichia coli-Saccharomyces cerevisiae shuttle vector. This vector was shown to direct the expression in Saccharomyces cerevisiae of correctly processed, mature HSA which was recognized by antiserum to HSA, and possessed the correct N-terminal amino acid sequence.
Synthetic genes coding for elongated precursors of the vasoactive intestinal polypeptide (VIP) were synthesized and cloned in a highly efficient Escherichia coli expression vector. The synthetic ...genes code for VIP with its methionine (at position 17) replaced by leucine and elongated at the C‐terminus by Gly (vasoactive intestinal polypeptide‐Gly, i.e. VIPa) or by Gly‐Lys‐Arg (vasoactive intestinal polypeptide‐Gly‐Lys‐Arg, i.e. VIPb). The synthetic genes fused to the N‐terminal part of the E. coliβ‐galactosidase gene were expressed to yield high amounts of fusion proteins reaching upon induction at least 60% of the total cellular protein. The fusion proteins of 314 and 316 amino acids carrying in their C‐terminal portion either the 29 or 31 amino acids long VIP precursor polypeptide were shown to be immunoreactive with VIP antisera and were further purified and cleaved by CNBr. The resulting purified peptide precursors (VIPa and VIPb) were recognized by VIP receptors in rat liver plasma membranes and by antibodies to porcine VIP in a radioimmunoassay. Both precursors activated adenylate cyclase in rat liver membranes and stimulated pancreatic secretion in the cat. The affinity and potency of the cloned precursors is close to that of VIP purified from porcine intestine, suggesting that the elongated VIP precursors may act even without being converted into the C‐terminal amide form of the peptide. The elongated VIP precursors expressed in E. coli may provide a cheap, large‐scale source of experimental material for studies on VIP actions.
A general method to convert single-stranded, chemically synthesized oligonucleotides into cloned duplexes is described. Oligonucleotides supplied with 3'-terminal extensions that are complementary to ...3'-protruding ends obtained by certain restriction enzymes can be cloned either directly or with the help of an adapter molecule into double-stranded vectors. Two methods have also been developed for consecutive cloning applications. According to these methods, the synthetic oligonucleotides (and their enzymatically prepared complementary strands) are joined, one after the other, inside a cloning vector, each joining requiring one cloning step. Synthetic genes are thus built up from oligonucleotides corresponding to only one strand of the DNA. The sequential assembly of the cloned duplex takes place in the 5' to 3' direction. Each oligonucleotide is supplied with a four-nucleotide-long 3'-terminal extension, but this sequence is eliminated when the joining takes place, leaving no limiting sequence between the oligonucleotides. The two consecutive cloning methods, the adapter and the polycloning site methods, are illustrated by the assembly of short artificial genes.
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