Use of transformation to make targeted sequence alterations at the am (GDH) locus of Neurospora

Summary Specific in vitro-generated insertion, replacement, and deletion mutations have been integrated near the chromosomal locus ofam (NADP-specific glutamate dehydrogenase) ofNeurospora crassa. Two approaches have been successful. One approach usedam ⁺-containing vectors capable of integrating at any site in the genome. This technique was used to introduce a specific 700 bp insertion near theam locus and to replace chromosomal sequences nearam with plasmid DNA. Efficiency was low, however, and many transformants had to be screened to find the desired alterations among the ectopic insertions unless the incoming DNA had a large region of homology with theam region. A second approach increased the efficiency by using vectors containing a truncatedam gene, so that prototrophs could arise only by homologous recombination. Overall transformation frequency was reduced relative to the first method, but a large fraction of the transformations involved specific alterations of theam region.     

Nucleotide sequence of the glutamine synthetase gene (glnA) and its upstream region from Bacillus cereus

We have determined the complete nucleotide sequence of a 2.4 kb chromosomal EcoT22I-NspV fragment, containing the Bacillus cereus glnA gene (structural gene of glutamine synthetase). The deduced amino acid sequence indicates that the glutamine synthetase subunit consists of 444 amino acid residues (50,063 Da). Comparisons are made with reported amino acid sequences of glutamine synthetases from other bacteria. Upstrem of glnA we found an open reading frame of 129 codons (ORF129) preceded by the consensus sequence for a typical promoter. Maxicell experiments showed two polypeptide bands, with molecular weights in good agreement with that of glutamine synthetase and that of ORF129, in addition to vector-coded protein. It is possible that the product of this open reading frame upstream of glnA has a regulatory role in glutamine synthetase expression.     

Nucleotide sequence of the yeast regulatory gene GAL80

The GAL80 gene in Saccharomyces cerevisiae encodes a negative regulatory protein for the set of inducible genes involving metabolism of galactose and melibiose. We have determined the nucleotide sequence of GAL80 and its flanking regions and assigned the 5' end of its mRNA to the sequence. The deduced coding sequence for GAL80 protein contains 1305 nucleotides and the calculated molecular weight of the peptide chain is 48309. The 5' end of the GAL80 mRNA maps about 67 nucleotides upstream from the translation initiating ATG. We have also determined the nucleotide sequence of uninducible alleles GAL80S-0, GAL80S-1 and GAL80S-2, and found single base substitution in each of these mutant genes which would lead to alteration of amino acid in GAL80 protein.     

Nucleotide sequence of the GDH gene coding for the NADP-specific glutamate dehydrogenase of Saccharomyces cerevisiae

The isolation of the Saccharomyces cerevisiae gene for NADP-dependent glutamate dehydrogenase (NADP-GDH) by cross hybridization to the Neurospora crassa am gene, known to encode for NADP-GDH is described. Two DNA fragments selected from a yeast genomic library in phage lambda gt11 were shown by restriction analysis to share 2.5 kb of common sequence. A yeast shuttle vector (CV13) carrying either to the cloned fragments complements the gdh- strain of S. cerevisiae and directs substantial overproduction of NADP-GDH. One of the cloned fragments was sequenced, and the deduced amino acid (aa) sequence of the yeast NADP-GDH is 64% homologous to N. crassa, 51% to Escherichia coli and 24% to bovine NADP-GDHs.     

Nucleotide sequence of the gene encoding adenovirus type 2 DNA binding protein.

We have determined the nucleotide sequence of the gene encoding adenovirus type 2 (Ad2) DNA binding protein (DBP). From the nucleotide sequence the complete amino acid sequence of Ad2 DBP has been deduced. A comparison of the amino acid sequences of Ad2 and Ad5 DBP, both 529 residues long, reveals that the C-terminal 354 residues of both sequences are identical. Within the N-terminal 175 amino acid residues Ad2 and Ad5 show nine differences. The site of mutation in Ad2 ND1ts23, a mutant with a temperature-sensitive DNA replication, was mapped at the nucleotide level. A single nucleotide alteration in the DBP gene, resulting in a leucine leads to phenylalanine substitution at position 282 in the amino acid sequence is responsible for the temperature-sensitive character of this mutant. Previously, we localized the mutation of another DBP mutant with a temperature-sensitive DNA replication (H5ts125) at position 413 in the amino acid sequence of the DBP molecule (Nucleic Acids Res. 9 (1981) 4439-4457). These mapping data are discussed in relation to the structure and function of the DBP molecule.