Cloning and analysis of beta-tubulin gene from a protoctist.

We have isolated and characterized by restriction endonuclease mapping, transcription pattern, and DNA sequencing a beta-tubulin gene from the coenocytic freshwater protoctist, Achlya klebsiana. The gene is intronless and has a single open reading frame that encodes a 444-amino acid residue polypeptide of Mr 49,856. The protein shows a high degree of homology to other beta-tubulins, 85% identity to human beta-tubulin and 89% identity to beta-tubulin of the sporozoan (also a protoctist) Plasmodium falciparum. Fungal beta-tubulins are among the least identical to A. klebsiana beta-tubulin. Through Southern blot hybridization analysis, we determined that there is just one form of beta-tubulin gene in A. klebsiana. Transcription of the gene was studied during sporogenesis. Following induction of sporogenesis, the level of the mRNA increased markedly at 2 h and declined in the next 2 h when mitosis, cytokinesis, and spore development occurred. At the same time, beta-tubulin content increased about 6-fold in the cells. Sporulation in A. klebsiana is not inhibited by antimitotic drugs such as benomyl, colcemid, and colchicine. Benomyl resistance in Neurospora crassa and Aspergillus nidulans has been genetically and molecularly linked to single amino acid substitutions at positions 167 and 165, respectively. The change from phenylalanine to tyrosine conferring benomyl resistance to N. crassa is seen in A. klebsiana, but the valine substitution for alanine in A. nidulans is marked by cysteine replacement in A. klebsiana. The amino acid found at position 165 is not conserved in various beta-tubulins, but phenylalanine at position 167 is extremely conserved.

Molecular characterization of an NAD-specific glutamate dehydrogenase gene inducible by L-glutamine. Antisense gene pair arrangement with L-glutamine-inducible heat shock 70-like protein gene.

The gene for an NAD-specific glutamate dehydrogenase (NAD-GDH) that is allosterically activated by NADP+ (non-substrate) was cloned, and its physical structure and nucleotide sequence was determined. The gene consists of 9 introns and 10 exons; the 10th and largest exon, which is 1863 nucleotides long, is at the 3'-end of the gene. The shortest exon of 33 base pairs is the first and is located at the 5'-end of the gene. The large exon is in perfect register along the complementary strand with a heat shock 70 (HSP)-like protein gene. The NAD-GDH gene is inducible with L-glutamine, just as the HSP 70-like protein gene (LéJohn, H.B., Cameron, L.E., Yang, B., MacBeath, G., Barker, D.S., and Williams, S.A. (1994) J. Biol. Chem. 269, 4513-4522). The phenomenon of anti-parallel coupling of two genes is named antisense gene pair. By Northern and Western blotting techniques, we obtained indirect evidence that the gene is expressed in vivo. The gene encodes a protein of M(r) 118,740 which consists of 1063 amino acid residues. The 5' and 3' borders of the gene display typical but unproven promoter motifs of CCAAT, TATAAT, and AAATAAAA polyadenylation signal bounded by a pyrimidine-rich transcription termination-type format. Restriction endonuclease site mapping of all the genomic clones isolated that carry most or all of the gene, and of the genome itself, gave hybridization patterns that are consistent with the interpretation that the organism, Achlya klebsiana, has only one form of the gene. 3'-End-labeling of a 5.2-kb XbaI DNA fragment (carrying the antisense gene pair) that was then asymmetrically cleaved to produce two single 3'-end-labeled pieces that were used as probes on L-glutamine-induced cell poly(A)+ RNA, showed that the end-labeled DNA equivalent to the HSP 70-like protein mRNA hybridized to a 3.4-kb transcript and the end-labeled DNA equivalent to the NAD-GDH mRNA hybridized to a 2.4-kb transcript.

Cloning and analysis of a constitutive heat shock (cognate) protein 70 gene inducible by L-glutamine.

An intronless gene encoding a protein of 652 amino acid residues with an M(r) of 71,266, showing between 79% and 59% identity in nucleotide sequence with heat shock protein 70 (HSP 70) genes of Bremia lactucae (a parasitic Oomycete of lettuce) and a wide range of organisms that include humans, was isolated from the nonparasitic Oomycete Achlya klebsiana. While the gene appears to be constitutively expressed, L-glutamine augmented its expression particularly under conditions of nutritional stress. L-Glutamine enhanced the transcription of a 2.4-kilobase poly(A)+ RNA simultaneously in the same way as it elevated the cellular level of the HSP 70-like protein. A polyclonal antibody (affinity-purified) raised in rabbit against the purified monomeric (M(r) 120,000) form of an NAD-specific glutamate dehydrogenase (Yang, B., and LéJohn, H.B. (1994) J. Biol. Chem. 269, 4506-4512) immunoprecipitated the HSP 70-like protein, and it was used to study the kinetics of induction of this stress-related protein and the effect of proteinase inhibitors on its metabolism. By using as probes four partial length cDNA clones, nine overlapping DNA fragments of the organism's genome carrying the HSP 70-like protein gene were isolated from a genomic library. The nucleotide sequence of the gene, including its boundaries, was determined by using these genomic clones. The 5'-untranslated boundary of the gene displayed the classical nucleotide arrangement of heat shock elements as well as CCAAT and TATA box motifs. Within the coding region are the typical conserved amino acid heat shock protein signatures 1 and 2 at the predicted locations. By primer extension and S1 nuclease protection mapping system, we estimated that the gene is probably transcribed into a message of 2.2 kilobases.