Cloning of a cDNA from Arabidopsis thaliana homologous to the human XPB gene.

The human gene XPB, defective in xeroderma pigmentosum patients complementation group B, encodes a DNA helicase involved in several DNA metabolic pathways, including DNA repair and transcription. The high conservation of this gene has allowed the cloning of homologs in various species, such as mouse, yeast and Drosophila. Not much information on the molecular basis of nucleotide excision repair in plants is available, but these organisms may have similar mechanisms to other eukaryotes. A homolog of XPB was isolated in Arabidopsis thaliana by using polymerase chain reaction (PCR) with degenerate oligonucleotides based on protein domains which are conserved among several species. Screening of an Arabidopsis cDNA library led to the identification and isolation of a cDNA clone with 2670 bp encoding a predicted protein of 767 amino acids, denoted araXPB. Genomic analysis indicated that this is a nuclear single copy gene in plant cells. Northern blot with the cDNA probe revealed a major transcript which migrated at approx. 2,800 b, in agreement with the size of the cDNA isolated. The araXPB protein shares approximately 50% identical and 70% conserved amino acids with the yeast and human homologs. The plant protein maintains all the functional domains found in the other proteins, including nuclear localization signal, DNA-binding domain and helicase motifs, suggesting that it might also act as part of the RNA transcription apparatus, as well as nucleotide excision repair in plant cells.

Dual role for the yeast THI4 gene in thiamine biosynthesis and DNA damage tolerance.

The THI4 gene of Saccharomyces cerevisiae encodes an enzyme of the thiamine biosynthetic pathway. The plant homolog thi1, from Arabidopsis thaliana, is also involved in thiamine biosynthesis; but was originally cloned due to its capacity to complement DNA repair deficient phenotypes in Escherichia coli. Here, the behavior of a thi4 disrupted strain was examined for increased sensitivity to treatment with the DNA damaging agents ultraviolet radiation (UV, 254 nm) and methyl methanesulfonate (MMS). Although the thi4 null mutant showed a similar level of survival as the wild-type strain, a higher frequency of respiratory mutants was induced by the two treatments. A similar phenotype was seen with wild-type strains expressing an antisense THI4 construct. Further analysis of respiratory mutants revealed that these were due to mutations of mitochondrial DNA (mtDNA) rather than nuclear DNA, consisting of rho-petite mutants. Moreover, the frequency of mutations was unaffected by the presence or absence of thiamine in the growth medium, and the defect leading to induction of petites in the thi4 mutant was corrected by expression of the Arabidopsis thi1 gene. Thus, Thi4 and its plant homolog appear to be dual functional proteins with roles in thiamine biosynthesis and mitochondrial DNA damage tolerance.

Thi1, a thiamine biosynthetic gene in Arabidopsis thaliana, complements bacterial defects in DNA repair.

An Arabidopsis thaliana cDNA was isolated by complementation of the Escherichia coli mutant strain BW535 (xth, nfo, nth), which is defective in DNA base excision repair pathways. This cDNA partially complements the methyl methane sulfonate (MMS) sensitive phenotype of BW535. It also partially corrects the UV-sensitive phenotype of E. coli AB1886 (uvrA) and restores its ability to reactivate UV-irradiated lambda phage. It has an insert of ca. 1.3 kb with an open reading frame of 1047 bp (predicting a protein with a molecular mass of 36 kDa). This cDNA presents a high homology to a stress related gene from two species of Fusarium (sti35) and to genes whose products participate in the thiamine biosynthesis pathway, THI4, from Saccharomyces cerevisiae and nmt2 from Schizosaccharomyces pombe. The Arabidopsis predicted polypeptide has homology to several protein motifs: amino-terminal chloroplast transit peptide, dinucleotide binding site, DNA binding and bacterial DNA polymerases. The auxotrophy for thiamine in the yeast thi4::URA3 disruption strain is complemented by the Arabidopsis gene. Thus, the cloned gene, named thi1, is likely to function in the biosynthesis of thiamine in plants. The data presented in this work indicate that thi1 may also be involved in DNA damage tolerance in plant cells.

The Saccharomyces cerevisiae HSP12 gene is activated by the high-osmolarity glycerol pathway and negatively regulated by protein kinase A.

The HSP12 gene encodes one of the two major small heat shock proteins of Saccharomyces cerevisiae. Hsp12 accumulates massively in yeast cells exposed to heat shock, osmostress, oxidative stress, and high concentrations of alcohol as well as in early-stationary-phase cells. We have cloned an extended 5'-flanking region of the HSP12 gene in order to identify cis-acting elements involved in regulation of this highly expressed stress gene. A detailed analysis of the HSP12 promoter region revealed that five repeats of the stress-responsive CCCCT motif (stress-responsive element [STRE]) are essential to confer wild-type induced levels on a reporter gene upon osmostress, heat shock, and entry into stationary phase. Disruption of the HOG1 and PBS2 genes leads to a dramatic decrease of the HSP12 inducibility in osmostressed cells, whereas overproduction of Hog1 produces a fivefold increase in wild-type induced levels upon a shift to a high salt concentration. On the other hand, mutations resulting in high protein kinase A (PKA) activity reduce or abolish the accumulation of the HSP12 mRNA in stressed cells. Conversely, mutants containing defective PKA catalytic subunits exhibit high basal levels of HSP12 mRNA. Taken together, these results suggest that HSP12 is a target of the high-osmolarity glycerol (HOG) response pathway under negative control of the Ras-PKA pathway. Furthermore, they confirm earlier observations that STRE-like sequences are responsive to a broad range of stresses and that the HOG and Ras-PKA pathways have antagonistic effects upon CCCCT-driven transcription.

Induction of major heat-shock proteins of Saccharomyces cerevisiae, including plasma membrane Hsp30, by ethanol levels above a critical threshold.

Many of the changes induced in yeast by sublethal yet stressful amounts of ethanol are the same as those resulting from sublethal heat stress. They include an inhibition of fermentation, increased induction of petites and stimulation of plasma membrane ATPase activity. Ethanol, at concentrations (4-10%, v/v) that affect growth and fermentation rates, is also a potent inducer of heat-shock proteins including those members of the Hsp70 protein family induced by heat shock. This induction occurs above a threshold level of about 4% ethanol, although different heat-shock proteins and heat-shock gene promoters are optimally induced at different higher ethanol levels. In addition ethanol (6-8%) causes the same two major changes to integral plasma-membrane protein composition that result from a sublethal heat stress, reduction in levels of the plasma membrane ATPase protein and acquisition of the plasma membrane heat-shock protein Hsp30.

Regulation of THI4 (MOL1), a thiamine-biosynthetic gene of Saccharomyces cerevisiae.

THI4, a Saccharomyces cerevisiae gene originally identified as a result of transient expression in molasses medium and named MOL1 is regulated by thiamine. Using a THI4 promoter-lacZ fusion on a centromeric yeast vector, we have shown that the THI4 is completely repressed throughout batch culture by thiamine at a concentration around 1 microM, but shows high level constitutive expression in thiamine-free medium. The transient expression pattern observed in molasses medium can be mimicked by the addition of 0.15 microM-thiamine to defined minimal medium. Cells grown in thiamine-free medium have an intracellular thiamine concentration of around 9 pmol/10(7) cells. A low level (1 microM) of exogenous thiamine is completely sequestered from the medium within 30 min; intracellular thiamine concentrations rise rapidly, followed by a gradual decrease as a result of dilution during growth. A saturating extracellular level of thiamine leads to a maximal intracellular concentration of around 1600 pmol/10(7) cells, at which point the transport system is shut down. After transfer from repressing to non-repressing medium, THI4 becomes induced when the intracellular concentration of thiamine falls to 20 pmol/10(7) cells. A thi4::URA3 disruption strain is auxotrophic for thiamine, but can grow in the presence of hydroxyethyl thiazole, indicating that the gene product is involved in the biosynthetic pathway leading to the formation of the thiazole precursor of thiamine.

MOL1, a Saccharomyces cerevisiae gene that is highly expressed in early stationary phase during growth on molasses.

We have isolated a new Saccharomyces cerevisiae gene, MOL1, that is transiently expressed at high levels in the early stationary phase of batch cultures growing on industrial molasses medium. The DNA sequence of the MOL1 gene (for MOLasses-inducible) with its flanking regions was determined (EMBL accession number X61669). It encodes a polypeptide of M(r) 35 kDa that is closely related to stress-inducible proteins of similar size from two Fusarium species. Unlike ST135 of Fusarium, MOL1 is not induced by ethanol or heat shock. MOL1 expression is absent in rich (YP) medium, and only very low levels of expression are detectable in minimal (YNB) medium. The gene is not essential, and a MOL1 disruption strain showed no apparent phenotype under a variety of growth conditions. The 5' region of MOL1 contains the complete sequence previously determined for the SUF4 locus, encoding a tRNA-gly (UCC) gene, which has been mapped to chromosome VII.

HSP12, a new small heat shock gene of Saccharomyces cerevisiae: analysis of structure, regulation and function.

We have isolated a new small heat shock gene, HSP12, from Saccharomyces cerevisiae. It encodes a polypeptide of predicted Mr 12 kDa, with structural similarity to other small heat shock proteins. HSP12 gene expression is induced several hundred-fold by heat shock and on entry into stationary phase. HSP12 mRNA is undetectable during exponential growth in rich medium, but low levels are present when cells are grown in minimal medium. Analysis of HSP12 expression in mutants affected in cAMP-dependent protein phosphorylation suggests that the gene is regulated by cAMP as well as heat shock. A disruption of the HSP12 coding region results in the loss of an abundant 14.4 kDa protein present in heat shocked and stationary phase cells. It also leads to the induction of the heat shock response under conditions normally associated with low-level HSP12 expression. The HSP12 disruption has no observable effect on growth at various temperatures, nor on the ability to acquire thermotolerance.

Molecular analysis of actinidin, the cysteine proteinase of Actinidia chinensis.

We have isolated and sequenced two very similar cDNA clones of 1145 and 809 bp length, from a fruit-specific library of Actinidia chinensis, the larger encoding all 220 amino acids of actinidin, showing 91% homology to the published amino acid sequence. Both cDNAs code for an additional 25 amino acids following the mature carboxy terminus of actinidin. The larger clone has coding potential for 57 residues of an amino-terminal extension with considerable homology to amino-terminal sequences of other cysteine proteinases. From size determination of both mRNA (1.4 kb) and immunoprecipitated in vitro translation product (39 kDa) it was estimated that actinidin is synthesised as a precursor approximately 15 kDa larger than the mature protein. Both proteolytic cleavage sites are located on the surface of the molecule as illustrated by the hydropathy profile of the deduced amino acid sequence. Features of the prosegment primary sequence are considered with regard to a possible mechanism of inactivation of the proteinase, by analogy with other proteolytic zymogens. The presence of three potential glycosylation sites, one within the carboxy-terminal and two in the amino-terminal extension, are consistent with subcellular location of the enzyme within membrane-bound organelles. Results from a Southern blot suggest that actinidin is encoded by a multigene family of up to ten members. Actinidin gene expression, both at the level of mRNA and protein, is largely restricted to the fruit of the plant, where the level of actinidin mRNA accumulates early during development.