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P Klaassen

Publications and source records attributed to P Klaassen.

15 recordsLinked to original sources
PubMed1999

Glucose uptake kinetics and transcription of HXT genes in chemostat cultures of Saccharomyces cerevisiae.

The kinetics of glucose transport and the transcription of all 20 members of the HXT hexose transporter gene family were studied in relation to the steady state in situ carbon metabolism of Saccharomyces cerevisiae CEN.PK113-7D grown in chemostat cultures. Cells were cultivated at a dilution rate of 0.10 h-1 under various nutrient-limited conditions (anaerobically glucose- or nitrogen-limited or aerobically glucose-, galactose-, fructose-, ethanol-, or nitrogen-limited), or at dilution rates ranging between 0.05 and 0.38 h-1 in aerobic glucose-limited cultures. Transcription of HXT1-HXT7 was correlated with the extracellular glucose concentration in the cultures. Transcription of GAL2, encoding the galactose transporter, was only detected in galactose-limited cultures. SNF3 and RGT2, two members of the HXT family that encode glucose sensors, were transcribed at low levels. HXT8-HXT17 transcripts were detected at very low levels. A consistent relationship was observed between the expression of individual HXT genes and the glucose transport kinetics determined from zero-trans influx of 14C-glucose during 5 s. This relationship was in broad agreement with the transport kinetics of Hxt1-Hxt7 and Gal2 deduced in previous studies on single-HXT strains. At lower dilution rates the glucose transport capacity estimated from zero-trans influx experiments and the residual glucose concentration exceeded the measured in situ glucose consumption rate. At high dilution rates, however, the estimated glucose transport capacity was too low to account for the in situ glucose consumption rate.

Biological Transport↗
PubMed1992

Characterization of the 56-kDa subunit of yeast trehalose-6-phosphate synthase and cloning of its gene reveal its identity with the product of CIF1, a regulator of carbon catabolite inactivation.

Trehalose-6-phosphate synthase is the key enzyme for biosynthesis of trehalose, the major soluble carbohydrate in resting cells of yeast. This enzyme was purified from a strain of Saccharomyces cerevisiae lacking vacuolar proteases. It was found to be a multimeric protein of 630 kDa. Monoclonal antibodies were raised against its smallest subunit (56 kDa) and used for screening a yeast cDNA library. This yielded an immunopositive cDNA clone of 1.7 kb, containing an open reading frame of 1485 base pairs. Its sequence, called TPS1 (for trehalose-6-phosphate synthase), was represented by a single gene in the yeast genome and was found to be almost identical with the recently sequenced CIF1, a gene important for carbon catabolite inactivation, believed to be allelic with FDP1. A mutant obtained by disruption of TPS1 had a very low activity of trehalose-6-phosphate synthase, indicating that TPS1 is an important component of the enzyme. The mutant also showed a growth defect when transferred from glycerol to glucose, a phenotype similar to that of the cif1 and fdp1 mutants deficient in carbon catabolite inactivation. Thus, the smallest subunit of the biosynthetic enzyme trehalose-6-phosphate synthase appears to have, in addition, a central regulatory role in the carbohydrate metabolism of yeast.

Amino Acid Sequence↗
PubMed1989

The prostate-specific antigen gene and the human glandular kallikrein-1 gene are tandemly located on chromosome 19.

Using a prostate-specific antigen cDNA as a hybridization probe, clones containing the kallikrein genes encoding prostate-specific antigen, human glandular kallikrein-1 and pancreas/kidney kallikrein were isolated from a human genomic library. Clones containing the prostate-specific antigen gene and the human glandular kallikrein-1 gene overlap and span a region of about 36 kb. The two genes are aligned in a head to tail orientation at a mutual distance of 12 kb. Southern blot analysis of DNA from a panel of human-hamster hybrid cells with specific probes revealed the genes to be situated on chromosome 19. Assuming that the pancreas/kidney kallikrein gene is located in the same cluster, the distance to the prostate-specific antigen gene and the human glandular kallikrein gene must be at least 15 kb.

Animals↗
PubMed1989

The human androgen receptor: domain structure, genomic organization and regulation of expression.

The domain structure and the genomic organization of the human androgen receptor (hAR) has been studied after molecular cloning and characterization of cDNA and genomic DNA encoding the hAR. The cDNA sequence reveals an open reading frame of 2751 nucleotides encoding a protein of 917 amino acids with a calculated molecular mass of 98,845 D. The N-terminal region of the hAR is characterized by a high content of acidic amino acid residues and by several homopolymeric amino acid stretches. The DNA-binding domain showed a high homology with the DNA-binding domain of the human glucocorticoid receptor (hGR) and the human progesterone receptor (hPR). The predominantly hydrophobic steroid binding domain of the hAR is 50-55% homologous with the ligand binding domains of the hGR and hPR. Transient expression of recombinant AR cDNA in COS-cells resulted in the production of a 110 kDa protein with the expected binding specificity of androgen receptors. Co-transfection with a reporter-gene construct [CAT(chloramphenicol acetyl transferase) under direction of the androgen regulated MMTV-promoter] showed that the protein is functionally active with respect to transcription regulation. In the LNCaP prostate carcinoma cell line two major (11 and 8 kb) and one minor (4.7 kb) mRNA species can be found which can be down-regulated by androgens. The hAR protein coding region was shown to be divided over eight exons with an organization similar to that of the progesterone and oestrogen receptor. The sequence encoding the N-terminal domain was found in one large exon. The two DNA-binding fingers were encoded by two small exons; the information for the androgen-binding domain was found to be distributed over five exons. Southern blot analysis of genomic DNA revealed that the hAR is encoded by one single gene, which is situated on the X-chromosome.

Animals↗
PubMed1989

Oncogene expression in Rauscher murine leukemia virus induced erythroid, myeloid and lymphoid cell lines.

A comparative study on the expression of nuclear and cytoplasmic oncogenes was carried out using the Northern blotting technique, in Rauscher virus induced primary leukemias and the more malignant transformed cell lines derived from them. The latter grow permanently in vitro. Hyperplastic spleens obtained from mice recovering from anemia were analysed as controls. In addition to the detection of mRNAs, Southern blotting was carried out to observe whether rearrangement or amplification of oncogenes had occurred. The results show that the nuclear oncogenes c-myc, c-myb and p53 are strongly expressed in leukemic tissue, whereas c-fos transcripts show a much weaker hybridization. The expression of two of these oncogenes, c-myc and c-myb was followed during differentiation in myeloid leukemic cells and showed a gradual decrease when compared with the actin gene, which is constitutively transcribed. A large number of cytoplasmic oncogenes is expressed in the leukemic cells lines, i.e. c-abl, c-fms, c-fes, c-src, c-ros, c-H-ras, c-K-ras and N-ras. Of these, transcripts coding for c-abl and c-src were absent in blast cells of acute erythroid leukemias. Transcripts coding for c-erb, c-mos and c-sis could also not be detected. A number of putative oncogenes which are reported to play a role in Moloney and Friend virus induced leukemias for instance pim-1, fis-1, fim-1 and fim-2 were also used for screening. Only expression of pim-1 in Rauscher virus induced myeloid leukemic cells and in primary acute erythroid leukemias could be observed. At the DNA level no rearrangement or amplification of any of the oncogenes investigated could be detected. The results show that a number of oncogenes are expressed simultaneously in the same leukemic tissue or cell lines. It therefore seems likely that the presence of transcripts of different oncogenes is associated with the progression of leukemia, but is not the primary cause of leukemogenesis or of the transformation of these cells into established cell lines.

Animals↗
PubMed1989

Structural organization of the human androgen receptor gene.

The complete coding region of the human androgen receptor gene has been isolated from a genomic library. The information for the androgen receptor was found to be divided over eight exons and the total length of the gene exceeded 90 kb. The sequence encoding the N-terminal region is present in one large exon. The two putative DNA-binding fingers are encoded separately by two small exons. The information for the hormone-binding domain is split over five exons. Positions of introns are identical to those reported for the chicken progesterone receptor and the human oestrogen receptor genes. Southern blot analysis of genomic DNA with various specific probes reveal that the human androgen receptor is encoded by a single-copy gene.

Amino Acid Sequence↗
PubMed1988

Molecular cloning and characterization of novel prostate antigen cDNA's.

Three different prostate antigen cDNA's were isolated from a PC 82 prostate tumor cDNA library. PA 75 has a size of 1.4 kb and contains the almost complete information for the 35 kD prostate antigen preproprotein. The 1.6 kb PA 525 cDNA lacks about 0.2 kb of the 3'-non coding region and contains an additional internal 0.4 kb fragment as a result of alternative splicing. PA 424 represents a 0.6 kb variant of PA 75. It contains a 0.15 kb internal fragment and a poly(A) tail preceded by an AAGAAA motive at the 3'-end. The predicted protein products of PA 525 and PA 424 will be different from PA 75 at the C-terminal end. In RNA preparations of two human prostate tumors (PC 82 and PC EW) seven different prostate antigen transcripts can be detected ranging in size from 0.5 kb to 5.6 kb. PA 75 cDNA represents the major 1.5 kb mRNA. PA 424 correlates with a 0.9 kb transcript and PA 525 with a 1.9 kb mRNA species.

Amino Acid Sequence↗
PubMed1988

Cloning, structure and expression of a cDNA encoding the human androgen receptor.

A cDNA clone has been isolated from a library prepared of mRNA of human breast cancer T47D cells with an oligonucleotide probe homologous to part of the region encoding the DNA-binding domain of steroid receptors. The clone has a size of 1505 bp and sequence analysis revealed an open reading frame of 1356 bp. The deduced amino acid sequence displays two highly conserved regions identified as the putative DNA-binding and hormone binding domains respectively of steroid receptors. Expression of this cDNA clone in COS cells produces a nuclear protein with all the binding characteristics of the human androgen receptor (hAR). The gene encoding the cDNA is assigned to the human X-chromosome. High levels of three hybridizing mRNA species of 11, 8.5 and 4.7 kb respectively are found in the human prostate cancer cell line (LNCaP), which contains elevated levels of hAR. The present data provide evidence that we have isolated a cDNA that encodes a major part of the human androgen receptor.

Amino Acid Sequence↗
PubMed1988

Effects of FSH and IGF-I on immature rat Sertoli cells: inhibin alpha- and beta-subunit mRNA levels and inhibin secretion.

Effects of follicle-stimulating hormone (FSH) and insulin-like growth factor-I (IGF-I) on inhibin production by cultured Sertoli cells from 21- to 23-day-old rats were studied. The expression of inhibin alpha- and beta-subunit mRNAs, and inhibin immunoreactivity and in vitro bioactivity were estimated. Using a cDNA probe corresponding to the alpha-subunit of bovine inhibin, specific hybridization with a 1.5-1.7 kilobase (kb)mRNA species was observed. Addition of FSH to the cultured Sertoli cells for 24 h markedly increased the level of this mRNA in a dose-dependent way. IGF-I had no effect on the intensity of the hybridization. Using a cDNA probe corresponding to the beta B-subunit of human inhibin, 3.5 and 4.2 kb mRNA species were detected. FSH and IGF-I had no effect on the hybridization signal. No hybridization was observed with a cDNA probe corresponding to the beta A bovine inhibin subunit. Inhibin activity was detected in cells and medium by immunoassay, and in the medium by in vitro bioassay. FSH stimulated both immunoreactivity and in vitro bioactivity, whereas IGF-I had no effect at all. The present effect of FSH on inhibin alpha-subunit mRNA expression in cultured Sertoli cells indicates that regulation of inhibin production by FSH includes an effect at the transcriptional level. However, this does not exclude additional translational and posttranslational effects.

Animals↗
PubMed1988

Structure-function analysis of murine interferon-alpha: antiviral properties of novel hybrid interferons.

As described earlier the protein products of the murine interferon (IFN) genes MuIFN-alpha 1, -alpha 2, and -alpha 4 differ in their antiviral activity on hamster (CHO) and mouse (L929) cells. For structure-function analysis, hybrids were prepared between the three genes using common restriction enzyme sites. Natural and hybrid genes were transiently expressed in monkey COS cells. Under the conditions used IFN constituted 20-30% of the total amount of secreted proteins. Using a panel of hybrids either between alpha 1 and alpha 2 or between alpha 1, alpha 2, and alpha 4, the amino-terminal region of the protein, from amino acids 10 to 58, was found to determine its antiviral activity on hamster cells. On mouse cells, the antiviral activities of hybrids between alpha 4 and either alpha 1 or alpha 2 were compared. The high activity of alpha 4 (five to ten times that of alpha 1 or alpha 2) was not transmitted to hybrids having the amino-terminal part of alpha 4, but coincided with the presence of the alpha 4 carboxy-terminal region in all but one hybrid construct. The deletion of five amino acids (positions 103-107) located in this region of alpha 4 did not affect antiviral activity when introduced into MuIFN-alpha 2 and a MuIFN-alpha 42 hybrid by site-directed mutagenesis.

Animals↗
PubMed1988

Structure-function analysis of mouse interferon alpha species: MuIFN-alpha 10, a subspecies with low antiviral activity.

A mouse interferon alpha gene (MuIFN-alpha 10) was isolated from a BALB/c cosmid genomic library. The gene was located on a 1.8 kb HindIII fragment and a 5.1 kb EcoRI fragment. The coding region and parts of the 5' and 3' non-coding regions were sequenced. The results showed that the MuIFN-alpha 10 gene encoded a protein of 167 amino acids. Like most other MuIFN-alpha species it contained a putative N-glycosylation site at amino acid positions 78 to 80. It also possessed cysteine residues at positions 1, 29, 86, 99 and 129. In the signal peptide, in addition to cysteine 21, which is present in all MuIFN-alpha species sequenced so far, a cysteine was found at position 22. At the amino acid level MuIFN-alpha 10 showed strong homology to MuIFN-alpha 1 (only 15 out of 167 amino acids were different). The MuIFN-alpha 10 gene was transiently expressed in monkey COS cells under the direction of the simian virus 40 early promoter. The protein product secreted by COS cells was equally active on mouse (L929) and hamster (CHO) cells. However, as compared to MuIFN-alpha 1 and MuIFN-alpha 4 the specific activity on mouse cells of the protein was 10- to 100-fold lower. To find out which region of its structure was responsible for this low activity, hybrids of the genes encoding MuIFN-alpha 10 and MuIFN-alpha 1 were constructed using the two common XmmI sites which correspond to positions between amino acids 67 and 68 and 123 and 124, respectively. The data showed that hybrid constructs which were MuIFN-alpha 1-like from amino acid 68 or MuIFN-alpha 10-like from position 124 to the C terminus possessed high antiviral activity. Other hybrid constructs were hardly active at all. This implied that the amino acid 68 to 123 region was mainly responsible for the low antiviral activity of MuIFN-alpha 10. In this part of the molecule MuIFN-alpha 1 and MuIFN-alpha 10 differed in only five amino acids. A serine at position 110 and a valine at 85 were unique to MuIFN-alpha 10 as compared to all known MuIFN-alpha and human IFN-alpha subspecies.

Amino Acid Sequence↗