We present a procedure for cosmid cloning that allows rapid and efficient cloning of individual DNA fragments of between 32kb and 45kb. By appropriate treatment of the cloning vector, pJb8, we make left-hand and right-hand vector ends that are incapable of self-ligation but which accept dephosporylated insert DNA fragments. The inserted fragments are generated by partial digestion with MboI or Sau3A and are dephosphorylated to prevent ligation and insertion of non-contiguous fragments. The method eliminates the need to size the insert DNA fragments and prevents formation of clones containing short or multiple inserts. 1 microgram of target Drosophila DNA gives about 5 x 10(5) clones, with an average insert size of 38kb. We also describe a rapid and efficient method for preparing plasmid and cosmid DNA.

The mating‐type region of fission yeast consists of three components, mat1, mat2‐P and mat3‐M, each separated by 15 kb. Cell‐type is determined by the alternate allele present at mat1, either P in an h+ or M in an h‐ cell. mat2‐P and mat3‐M serve as donors of information that is transposed to mat1 during a switch of mating type. We have determined the nucleotide sequence of each component of mat. The P and M specific regions are 1104 and 1128 bp, respectively, and bounded by sequences common to each mating‐type cassette (H1; 59 bp and H2; 135 bp). A third sequence is present at mat2‐P and mat3‐M but absent at mat1 (H3; 57 bp), and may be involved in transcriptional repression of these cassettes. mat1‐P and mat1‐M each encode two genes (Pc; 118 amino acids, Pi; 159 amino acids, Mc; 181 amino acids and Mi; 42 amino acids). Introduction of opal or frame‐shift mutations into the open‐reading‐frame of each gene revealed that Pc and Mc are necessary and sufficient for mating and confer an h+ or h‐ mating type respectively. All four genes are required for meiotic competence in an h+/h‐ diploid. The transcription of each mat gene is strongly influenced by nutritional conditions and full induction was observed only in nitrogen‐free medium. The predicted product of the Pi gene contains a region of homology with the homeobox sequence, suggesting that this gene encodes a DNA binding protein that directly regulates the expression of other genes.

Aminoglycoside antibiotics in Escherichia coli and yeast can cause ribosomes to read through stop codons during translation. This can result in the phenotypic suppression of nonsense mutations. We show here for the first time that the aminoglycosides G-418 and paromomycin have similar effects in monkey (COS-7) cells in vivo. Suppression of an amber mutation (TAG) by aminoglycosides can restore the activity of a mutant gene transfected into COS-7 cells to almost 20% of wild type levels.

No abstract

G protein-coupled receptors (GPCRs) are the largest family of genes in animal genomes and represent more than 2% of genes in humans and C. elegans. These evolutionarily conserved seven-transmembrane proteins transduce a diverse range of signals. In view of their pivotal role in cell signaling, it is perhaps surprising that decades of genetic analysis in C. elegans, and recent genome-wide RNAi screens, have identified very few GPCR mutants. Therefore, we screened all GPCRs predicted to bind either small-molecule neurotransmitters or neuropeptides by using RNAi and quantitative behavioral assays. This shows that C16D6.2, C25G6.5, C26F1.6, F35G8.1, F41E7.3, and F59C12.2 are likely to be involved in reproduction, whereas C15B12.5, C10C6.2, C24A8.4, F15A8.5, F59D12.1, T02E9.1, and T05A1.1 have a role in locomotion. Gene deletions for F35G8.1 and T05A1.1 resulted in the same phenotype as that seen with RNAi. As some GPCRs may be resistant to RNAi, or may result in abnormalities not screened for here, the actual proportion of nonredundant receptors with an assayable function is probably greater. Strikingly, most phenotypes were observed for NPY-like receptors that may bind neuropeptides. This is consistent with the known actions of neuropeptides on the body wall muscle and reproductive tract in nematodes.