The highly conserved Chinese hamster GNAI3 gene maps less than 60 kb from the AMPD2 gene and lacks the intronic U6 snRNA present in its human counterpart.

The identity of a gene coamplified with the adenylate deaminase 2 gene (AMPD2) in coformycin-resistant cells was determined by analysis of its genomic sequence. Sequence comparisons reveal a significant homology with the 3' terminal part of the gene encoding the alpha i3 subunit of Gi proteins from several species (GNAI3). Identification of the gene was confirmed by Western blot analysis of its products. A precise sequence comparison was performed with the human genomic sequence. It showed that conservation remains important in noncoding exons as well as in introns. However, sequences corresponding to combined U6 snRNA and E protein pseudogene, previously identified inside intron 7 of the human gene, were not found in the Chinese hamster gene. GNAI3 is mapped to a region of conserved linkage between human chromosome 1 (locus 1p13) and mouse chromosome 3 (at 48.4 cM). The Chinese hamster GNAI3 gene maps to chromosome 1 within a 120-kb fragment that also comprises the AMPD2 and GSTM genes.

Co-amplified markers alternate in megabase long chromosomal inverted repeats and cluster independently in interphase nuclei at early steps of mammalian gene amplification.

Two-colour in situ hybridization with probes for two co-amplified markers located several megabases apart on chromosome 1 has been used to analyse early stages of adenylate deaminase 2 (AMPD2) gene amplification in Chinese hamster cells. In the amplified chromosomal structures, the distribution of hybridization spots identifies megabase-long inverted repeats. Their organization is remarkably well accounted for if breakage-fusion-bridge cycles involving sister chromatids drive the amplification process at these early stages. During interphase the markers often segregate into distinct nuclear domains. Many nuclei have bulges or release micronuclei, carrying several copies of one or both markers. These observations indicate that the amplified units destabilize the nuclear organization and eventually lead to DNA breakage during interphase. We propose a model in which interphase breakage has a role in the progression of gene amplification.

SDC25, a CDC25-like gene which contains a RAS-activating domain and is a dispensable gene of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the CDC25 gene product activates adenylate cyclase through RAS1 and RAS2 gene products. We have recently described the cloning of a DNA fragment which suppresses the cdc25 mutation but not ras1, ras2, or cdc35 mutations. This fragment contains a 5'-truncated open reading frame which shares 47% identity with the C-terminal part of the CDC25 gene. We named the entire gene SDC25. In this paper, we report the cloning, sequencing, and characterization of the complete SDC25 gene. The SDC25 gene is located on the chromosome XII close to the centromere. It is transcribed into a 4-kb-long mRNA that contains an open reading frame of 1,251 codons. Homology with the CDC25 gene extends in the N-terminal part, although the degree of similarity is lower than in the C-terminal part. In contrast with the C-terminal part, the complete SDC25 gene was found not to suppress the CDC25 gene defect. A deletion in the N-terminal part restored the suppressing activity, a result which suggests the existence of a regulatory domain. The SDC25 gene was found to be dispensable for cell growth under usual conditions. No noticeable phenotype was found in the deleted strain.

Purification and carbohydrate structure of natural murine interferon-beta.

Mouse interferon-beta (Mu-INF-beta) induced in C-243 cells with Newcastle disease virus was purified in four steps including ammonium sulfate fractionation. DEAE-cellulose, monoclonal Mu-IFN-beta antibody affinity and Mono-S cation-exchange chromatographies. Specific activity of the purified Mu-IFN-beta ranged over 1.1-1.4 X 10(9) NIH units/mg protein. This preparation was submitted to pronase digestion and gel on Fractogel TSK HW-40. The permethylated and acetylated glycopeptide fraction was analyzed by chemical-ionization (ammonia) mass spectrometry. The major glycopeptide is composed of Gal, Man, GlcNAc and NeuAc with a molar ratio of 2.0:3.6:3.4:0.5. The GLC pattern of methyl derivatives obtained by methanolysis and acetylation of fully methylated glycopeptide identified 2,3,4,6-tetra-O-methylgalactose; 3,4,6-tri-O-methyl-mannose; 2,3,4- and 2,4,6-tri-O-methylgalactose; 2,4,di-O-methyl mannose and 3,6-di-O-methylglucosamine. These results when compared with data on N-glycans suggest the following structure for the carbohydrate moiety of Mu-INF-beta: (formula; see text).

Structure of a murine alpha interferon pseudogene with a repetitive R-type sequence in the 3' flanking region.

A murine alpha interferon pseudogene was identified in a mouse genomic library. The nucleotide sequence revealed several in-phase termination codons within the gene and repetitive oligonucleotides in the flanking regions. The nucleotide sequences and the amino acids of the peptide signal sequences were compared with known human alpha interferon genes and the pseudogene.

Structure of mouse interferon-beta cDNA clones: sequence rearrangements in a cloned cDNA.

A fraction enriched in interferon (IFN) mRNA was prepared from mouse C243-3 induced cells and was used for the construction of a cDNA library. Two plasmids were obtained after screening by differential colony hybridization and IFN mRNA hybridization-selection and translation. The nucleotide sequences of the cDNA inserts revealed that both were partial copies of IFN-beta mRNA. The cDNA 861 corresponds to the entire 3' nontranslated region of the mRNA while the cDNA 2939 consists of rearranged translated regions of IFN mRNA. A mechanism for the rearrangement events during cDNA synthesis is proposed. A chromosomal DNA fragment hybridizing to cDNA 2939 was identified by screening a mouse genomic library.

Substrate specificity and adenosine triphosphatase activity of the ATP-dependent deoxyribonuclease of Bacillus subtilis.

Studies on the specificity of the ATP-dependent DNase of Bacillus subtilis 168, carried out with pure enzyme at the optimal conditions for its action, have shown that the substrate is double-stranded linear DNA. Linear single-stranded DNA (separated strands of B. subtilis DNA and linear phage fd DNA) is not attacked, neither are there any circular forms (supercoiled or nicked simian virus 40 and circular single-stranded fd DNAs). The double-stranded DNA can be completely hydrolysed, the limit products being, almost exclusively, mononucleotides. The presence of terminal phosphate residues in the substrate (either at the 3' or the 5' end) is not necessary for enzyme action. This DNase appears therefore to be an exonuclease processively liberating mononucleotides from both strands of the native linear DNA. ATP (indispensable for the DNase reaction) is also hydrolysed by the enzyme, to ADP and inorganic orthophosphate (Pi) in the presence of DNA. The apparent Km for ATP, in the ATPase reaction, is 0.15 mM. At high ATP concentrations, which inhibit the DNase activity, there is activation of the ATPase reaction. Three molecules of ATP are consumed for each DNA phosphodiester bond split, at optimal conditions for DNase activity.