Novel alleles of yeast hexokinase PII with distinct effects on catalytic activity and catabolite repression of SUC2.

In the yeast Saccharomyces cerevisiae, glucose or fructose represses the expression of a large number of genes. The phosphorylation of glucose or fructose is catalysed by hexokinase PI (Hxk1), hexokinase PII (Hxk2) and a specific glucokinase (Glk1). The authors have shown previously that either Hxk1 or Hxk2 is sufficient for a rapid, sugar-induced disappearance of catabolite-repressible mRNAs (short-term catabolite repression).

During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1.

In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (= GGS1 = FDP1 = BYP1 = CIF1 = GLC6 = TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation.

The genomic structure of the murine alpha 4 integrin gene.

The VLA-4 (alpha 4 beta 1) integrin is a leukocyte glycoprotein involved in both cell-extracellular matrix and cell-cell interactions. We report here the cloning of the murine alpha 4 gene whose protein product is antigenically related to the human VLA-4 alpha chain. The alpha 4 m gene is about 75 kb long and consists of 28 exons, ranging in size from 46 bp (exon 13) to 437 bp (exon 1).

A regulatory element in the 5'UTR directs cell-specific expression of the mouse alpha 4 gene.

Transfection experiments showed that the mouse alpha 4 promoter contains a downstream element in its 5'UTR which is essential for efficient promoter activity. DNaseI footprinting and electrophoretic mobility shift assays (EMSA) demonstrated that the region from nt +113 to +148 can bind a cell type-specific factor (MIII-3) present in the alpha 4m expressing cell line L1210 but not in the non-expressing cell lines A9 and LMTK. Two consensus SP1 sites in this 5'UTR were recognised in L1210, A9 and LMTK, and with extracts from A9 and LMTK, an AP2-like protein was shown to bind a downstream AP2 site.

Stable expression of VLA-4 and increased maturation of the beta 1-integrin precursor after transfection of CHO cells with alpha 4m cDNA.

A full-length cDNA coding for the murine alpha 4 integrin subunit (alpha 4m) was transfected into CHO-K1 cells and cell lines that expressed VLA-4 at their surface as a result of the association of transfected alpha 4m with endogenous hamster beta 1 were selected. Functionality of the expressed alpha 4m beta 1 was shown by adhesion assays on VCAM-1 and antibody (anti-VCAM-1) inhibition. Pulse chase experiments indicated that transfection of the murine alpha 4 cDNA into CHO cells led to an increase in maturation and a decrease in degradation of the beta 1 precursor subunit compared to control CHO-K1 cells.

Cloning and characterization of the promoter region of the murine alpha-4 integrin subunit.

To study the differential expression of the murine VLA-4 (alpha 4 beta 1) integrin, the 5'-flanking region of the gene for the alpha subunit (alpha 4m) was isolated and a cDNA for alpha 4m was obtained with reverse transcriptase polymerase chain reaction (RT-PCR). The cDNA sequence contained a difference in the signal peptide region compared to the previously described cDNA (Neuhaus et al., 1991).

Nucleotide sequence analysis of IS427 and its target sites in Agrobacterium tumefaciens T37.

We have determined the nucleotide sequence of IS427, an insertion sequence from Agrobacterium tumefaciens T37, IS427 is 1271 bp long, contains 16-bp imperfect terminal inverted repeats, and generates a 2-bp target sequence duplication. It is present at three sites in the pTiT37 plasmid and is absent from the chromosome of A. tumefaciens T37.

Identification of insertion sequence element IS427 in pTiT37 plasmid DNA of an Agrobacterium tumefaciens T37 isolate.

The isolation and characterization of an insertion sequence (IS) element, IS427, from Agrobacterium tumefaciens T37 is described. IS427 is present in three nonidentical copies on the pTiT37 plasmid. The copy that was isolated through transposition on the entrapment vector pUCD800 contains at its ends a 16-bp imperfect inverted repeat and generates a 2-bp duplication of the target DNA.

Nucleotide sequence of an insertion sequence (IS) element identified in the T-DNA region of a spontaneous variant of the Ti-plasmid pTiT37.

We have identified and determined the nucleotide sequence of an IS element (IS136) of Agrobacterium tumefaciens. This is the first IS element isolated and sequenced from a nopaline type Ti-plasmid. Our IS element has 32/30 bp inverted repeats with 6 mismatches, is 1,313 bp long and generates 9 bp direct repeats upon integration.

Nucleotide sequence of the T-DNA region encoding transcripts 6a and 6b of the pTiT37 nopaline Ti plasmid.

The nucleotide sequence of the T-DNA region encoding transcripts 6a and 6b of the pTiT37 nopaline Ti plasmid has been determined. Analysis of this sequence allowed us to find two open reading frames in opposite orientation and separated by 482 bp. Comparison with previous published nucleotide sequences of the homologous regions of the pTiAch5 and the pTi15955 octopine Ti plasmids reveals that the coding sequences as well as the 5'- and 3' 3-untranslated regions of both genes are highly homologous.