Transcriptional and biochemical regulation of a novel Arabidopsis thaliana bifunctional aspartate kinase-homoserine dehydrogenase gene isolated by functional complementation of a yeast hom6 mutant

An aspartate kinase-homoserine dehydrogenase (AK-HSDH) cDNA of Arabidopsis thaliana has been cloned by functional complementation of a Saccharomyces cerevisiae strain mutated in its homoserine dehydrogenase (HSDH) gene (hom6). Two of the three isolated clones were also able to complement a mutant yeast aspartate kinase (AK) gene (hom3). Sequence analysis showed that the identified gene (akthr2), located on chromosome 4, is different from the previously cloned A. thaliana AK-HSDH gene (akthr1), and corresponds to a novel bifunctional AK-HSDH gene. Expression of the isolated akthr2 cDNA in a HSDH-less hom6 yeast mutant conferred threonine and methionine prototrophy to the cells. Cell-free extracts contained a threonine-sensitive HSDH activity with feedback properties of higher plant type. Correspondingly, cDNA expression in an AK-deficient hom3 yeast mutant resulted in threonine and methionine prototrophy and a threonine-sensitive AK activity was observed in cell-free extracts. These results confirm that akthr2 encodes a threonine-sensitive bifunctional enzyme. Transgenic Arabidopsis thaliana plants (containing a construct with the promoter region of akthr2 in front of the gus reporter gene) were generated to compare the expression pattern of the akthr2 gene with the pattern of akthr1 earlier described in tobacco. The two genes are simultaneously expressed in meristematic cells, leaves and stamens. The main differences between the two genes concern the time-restricted or absent expression of the akthr2 gene in the stem, the gynoecium and during seed formation, while akthr1 is less expressed in roots.     

Expression of an Arabidopsis potassium channel gene in guard cells.

The Arabidopsis thaliana KAT1 cDNA encodes a voltage-gated inward-rectifying K+ channel. A KAT1 genomic DNA clone was isolated and sequenced, and a 5' promoter and coding sequences containing eight introns were identified. Reporter gene analysis of transgenic plants containing the KAT1 promoter fused to bacterial beta-glucuronidase showed robust beta-glucuronidase activity primarily in guard cells.     

Cloning of Salt Tolerance-Related cDNAs from the Mangrove Plant Sesuvium portulacastrum L.

In an attempt to isolate and identify the target genes relevant to salt tolerance in a mangrove plant （Sesuvium portulacastrum L.）, a subtracted cDNA library was constructed via suppressive subtractive hybridization （SSH）, in which the poly（A）＋RNA isolated from salt-tolerant S. portulacastrum leaves was used as a tester, whereas the driver was poly（A）＋RNA, derived from salt-sensitive S. portulacastrum leaves. Screening of this subtracted cDNA library revealed five clones, of which the expression levels in the salt-tolerant plant were markedly higher than those observed in the salt-sensitive plant, indicating that these candidate clones may be involved in salt-tolerance pathways. Among the clones isolated, P66, P175, and P233 are novel because no significant similarity was obtained upon alignment with the GenBank database. Clone P89 demonstrated high homology with NADPH of Arabidopsis thaliana, whereas clone P152 was highly homologous with the gene encoding late embryogenesis abundant （LEA） protein of A. thaliana. The full-length gene of clone P152, with a predicated 344 amino acid residues, was shown to bear LEA-2 domains, a signature motif for proteins that have been enriched under salty and drought conditions. It is thus implied that clone P152 would be a salt-tolerance gene of S. portulacastrum. In addition, we have also developed a strategy for the extraction of total RNA from mangrove plants.     

Structure and Expression of a Heat-Shock Protein 83 Gene of Pharbitis nil

Four cDNA clones representing mRNAs whose levels were affected by a photoperiod that induces flowering in Pharbitis nil were isolated by a differential hybridization screening procedure. The level of mRNAs represented by three clones (12L, 15L, and 17L) increased following a photoperiod that induces flowering and that represented by the fourth clone (clone 27) increased under conditions in which flowering was inhibited. DNA sequence analysis showed that one cDNA, clone 17L, is homologous to members of the 83- to 90-kD heat-shock protein (hsp) gene family. The corresponding gene, hsp83A, was isolated and its DNA sequence was determined. hsp83A encodes a protein that exhibits 70% amino acid identity with Drosophila melanogaster HSP83. The P. nil hsp83A gene contains two introns within the coding region. hsp83A mRNA was not detectable in cotyledons of plants grown in continuous light, but its level increased transiently following a 14-h dark period and reached a maximum 2 h after the lights were turned on. A dramatic increase in the level of hsp83A mRNA was also found 2 h after an end-of-day dark treatment. Genomic Southern blot analysis demonstrated that the P. nil hsp83-90 gene family consists of at least six members, one of which appears to be constitutively expressed in the light.     

Isolation and characterization of a Brassica napus cDNA corresponding to a B-class floral development gene

B-class floral homeotic genes are required for the proper formation and identity of petals and stamens in dicot flowers. A partial cDNA clone encoding a B-class gene, BnAP3 (Brassica napus APETALA3), was isolated from a B. napus cDNA library derived from young inflorescence meristems. The 5' region of the cDNA was retrieved by RACE. The deduced amino acid sequence of the full-length clone exhibited high similarity to APETALA3 of Arabidopsis thaliana and functionally homologous proteins from other species. 5' RACE and Southern analysis suggests that BnAP3 has multiple alleles in B. napus. Expression analysis assayed by RT-PCR shows that BnAP3 is expressed in floral tissues, as well as non-floral tissues such as root and bract. Transformation of wild-type A. thaliana and B. napus plants with BnAP3 under the control of a promoter specific to reproductive organs converts carpels to stamens, while the expression of this construct in A. thaliana plants mutant for AP3 restores the development of third-whorl stamens in addition to directing a carpel to stamen conversion in the fourth whorl.     

Structure and expression of elongation factor 2 gene during development of Dictyostelium discoideum

A cDNA library constructed from poly(A)+ RNA isolated from Dictyostelium discoideum cells at 12 h of development was screened with the hamster elongation factor 2 (EF-2) cDNA. Several different cDNA clones which hybridized were isolated after a second screening. A cDNA clone representing the 5'-end of the mRNA was obtained by primer extension. By comparing the amino acid sequence deduced from the nucleotide sequences of these clones with that of hamster EF-2, we found enough homology between them to conclude that the isolated clones were complementary to the mRNA of D. discoideum EF-2. The N terminus which is the GTP-binding domain and the C-terminal half where it interacts with a ribosome showed a high degree of homology. The amino acid sequence of the carboxyl half includes that it contain a site of ADP-ribosylation by diphtheria toxin. From the Northern blotting analysis, the size of the mRNA was estimated to be 2.6 kilobases. The expression of the mRNA was high in vegetative cells, became maximal at the aggregation stage, and decreased thereafter through development. Upon differentiation of prespore and prestalk cells, the mRNA was highly enriched in the former over the latter. ADP-ribosylation assay of EF-2 protein by diphtheria toxin showed nearly the same developmental changes for the protein as the mRNA. However, prestalk cells were found to contain the same amount of the protein as prespore cells. The Southern blot analyses indicated that the gene encoding EF-2 is unique.     

Isolation of a polyubiquitin promoter and its expression in transgenic potato plants.

A polyubiquitin clone (ubi7) was isolated from a potato (Solanum tuberosum) genomic library using a copy-specific probe from a stress-induced ubiquitin cDNA. The genomic clone contained a 569-bp intron immediately 5' to the initiation codon for the first ubiquitin-coding unit. Two chimeric beta-glucuronidase (GUS) fusion transgenes were introduced into potato. The first contained GUS fused to a 1156-bp promoter fragment containing only 5' flanking and 5' untranslated sequences from ubi7. The second transgene contained GUS translationally fused to the carboxy terminus of the first ubiquitin-coding unit and thus included the intron present in the 5' untranslated region of the polyubiquitin gene. Both ubi7-GUS transgenes were activated by wounding in tuber tissue and in leaves by application of exogenous methyl jasmonate. They were also expressed constitutively in the potato tuber peel (outer 1-2 mm). Both transgenes were actively expressed in mature leaves. Exceptionally high levels of expression were observed in senescent leaves. Transgenic clones containing the ubi7 intron and the first ubiquitin-coding unit showed GUS expression levels at least 10 times higher than clones containing GUS fused to the intronless promoter.     

A phosphate-starvation inducible β-glucosidase gene (psr3.2) isolated from Arabidopsis thaliana is a member of a distinct subfamily of the BGA family

We have previously isolated a phosphate starvation-response (psr) cDNA clone, psr3.1, from Brassica nigra which encodes a -glucosidase. Southern blots of Arabidopsis thaliana genomic DNA probed with the psr3.1 cDNA indicated that this gene exists as a single locus. A genomic library of A. thaliana was screened at high stringency to isolate the corresponding genomic clone. The resultant clone was coined psr3.2 because of its sequence divergence from isolated psr3.1 cDNA clones. Northern blotting with probes derived from the coding region of the genomic clone showed that this gene is expressed at high levels in P_i-starved roots and the enhancement occurred within two days of growth in medium lacking P_i. The expression of this gene is repressed by heat shock and anaerobic conditions, and it is not significantly induced by high salinity, or by nitrogen or sulfur deprivation. Sequence analysis of the genomic clone revealed the existence of 13 exons interrupted by 12 AT-rich introns and it possessed a high homology with the B. nigra psr3.1 as well as various other -glucosidase genes from other species. Sequence similarity and divergence percentages between the deduced amino acid sequences of the psr3 clones and other -glycosidases suggests that they should be included along with two other Brassicaceae genes in a distinct subfamily of the BGA glycosidase gene family. The presence of an endoplasmic reticulum retention signal at the carboxy terminus indicates the likely cellular location of PSR3.2. The possible metabolic and regulatory roles of this enzyme during the P_i-starvation response are discussed.     

Isolation of three novel Drosophila melanogaster genes containing repetitive sequences rich in GCN triplets.

Several cDNA and genomic clones were isolated from Drosophila melanogaster gene libraries by hybridization with a region of a mammalian gene that contains a simple repetitive sequence of six GCN repeats. One of the cDNA clones, E6, was completely sequenced and it was shown that it contains a region of 16 GCN repeats; these repeats encode a polyalanine stretch within a long open reading frame. The sequencing of three different genomic clones (A, B, and D) revealed that all the isolated Drosophila clones are similar to one another in a short region containing variable numbers of the GCN repeat. The genomic clone B was found to be the genomic counterpart of the cDNA clone E6. The other genomic clones, A and D, also hybridize with Drosophila cDNA clones at high stringency. These results indicate that the short GCN repetitive sequences, which we have named ala, are found within transcribed regions of the Drosophila genome. These Drosophila genes containing the ala repeat do not show significant sequence similarity to any presently known gene; we have named these novel genes ala-A, ala-B, and ala-D. The cDNA clone from gene ala-B was named ala-E6.     

Molecular cloning of a membrane bound tyrosine-specific protein kinase from rat spleen

Tyrosine-specific protein phosphorylation is believed to play an important (though poorly understood) role in various cellular functions in many normal and malignant cells. In order to understand the function of tyrosine-specific protein kinases in normal cells, it is necessary, as an initial step, to identify genes (and proteins) for these enzymes. For this purpose cDNA libraries were constructed in plasmid vector pGEM-3Z and lambda gt11 using mRNA from rat spleen. From these cDNA libraries, cDNA clones coding for a src-related tyrosine-specific protein kinase were isolated. The largest clone (L115) was 1.94 kb in size. Various restriction fragments of this clone were subcloned in plasmid vector for sequencing. The complete nucleotide sequence of the largest clone showed an open reading frame coding for a protein of 503 amino acids. The presence of a glycine at position 2 and an arginine at position 7 indicated that this protein is likely to be acylated at glycine 2 and therefore associated with plasma membrane. This gene showed high homology to human and mouse hck and hence it is perhaps the rat homologue of hck. Moderate level of expression of this gene was observed only in the adult rat spleen and not in other tissues. These results suggest that this kinase gene is expressed in a tissue specific manner.     

Structure and Light-Induced Expression of a Small Heat-Shock Protein Gene of Pharbitis nil

To isolate genes that are regulated by a photoperiod that promotes flowering in Pharbitis nil, a cDNA library representing mRNA of induced cotyledons was screened by differential hybridization. The DNA sequence of one cDNA clone isolated by this approach, clone 12L, showed homology to plant small heat-shock protein (hsp) genes. P. nil genomic clones hybridizing to clone 12L were isolated, and the DNA sequences of two P. nil small hsp (shsp) genes, shsp-1 and shsp-2, were determined. The derived amino acid sequences of shsp-1 and shsp-2 showed maximum homology to the 17.9-kD soybean hsp, a member of the class II cytoplasmic hsps found in plants. A study of the expression of shsp-1 and shsp-2 genes by RNase protection assay indicated that shsp-1 is induced by photoperiod, by light treatment of dark-grown P. nil seedlings, and by heat shock, and that shsp-2 is induced only by heat shock. Analysis of the sequences of the nontranscribed region indicates that both genes contain multiple heat-shock elements. The shsp-1 gene, in addition, contains sequences homologous to the GT-1-binding site, which may play a role in its light-regulated expression.