Effect of demographic factors, urinary peak flow rates, and Boyarsky symptom scores on patient treatment choice in benign prostatic hyperplasia

Thiopurine methyltransferase (TPMT) is a genetically polymorphic enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine. This genetic polymorphism is an important factor responsible for individual variation in thiopurine drug response. A cDNA for TPMT has been cloned from T84 human colon carcinoma cells. Northern blot analysis of multiple human tissues was performed with the T84 human colon carcinoma TPMT cDNA open reading frame (ORF) as a probe as one step toward understanding the molecular basis for the TPMT genetic polymorphism. Three mRNA species (approximately 1.4, 2.0, and 3.6 kb in length) were present in all tissues studied, including liver. However, none of these mRNAs matched the length of the 2.7 kb T84 TPMT cDNA. Therefore, it was important to clone a TPMT cDNA from a human drug-metabolizing organ such as the liver to determine whether its sequence matched that of the cDNA cloned from the T84 cell line. A human liver cDNA library was screened with the T84 TPMT cDNA ORF as a probe, and a 1.8 kb cDNA was isolated with a coding region sequence identical to that of the T84 TPMT cDNA. The TPMT cDNA ORF was then used to screen a human lymphocytes genomic DNA library in an attempt to clone the TPMT gene(s) in humans. Three intronless clones were isolated with identical ORF sequences that were 96% identical to that of the TPMT cDNA, but which contained multiple nucleotide substitutions and one deletion. The 3'- and 5'-flanking regions of one of the genomic DNA clones were sequenced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and expression of human Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase

Here we report the cDNA sequence of human Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase. The cDNA was isolated from a human placental cDNA library by screening with a 150bp probe generated by PCR using degenerate primers based on the sequences found in the sialyl motif. Comparative analysis of this cDNA with the rat liver alpha 2,3-sialyltransferase sequence indicates 91% nucleotide similarity between the two sequence in the predicted coding region. On the amino acid level, the degree of conservation is 97%. Surprisingly, Northern analysis indicated that the gene is expressed at low levels in human placenta but is abundantly expressed in skeletal muscle and fetal tissues.     

Expression of structure-specific recognition protein mRNA in fetal kidney and Fe-nitrilotriacetate-induced renal carcinoma in the rat

Specific expression of the structure-specific recognition protein (SSRP) gene was investigated in rat fetal, adult, and tumor tissues using a 2.0-kb partial sequence of rat SSRP cDNA isolated from a cDNA library of rat renal cell carcinoma. The results revealed that it was rather specifically expressed in rat fetal kidney and renal cell carcinoma induced by Fenitrilotriacetate, but not in adult kidney, when various organs were tested by Northern blot analysis. In situ hybridization further demonstrated that it was located in the neoplastic cells of renal cell carcinoma and in the epithelial cells of fetal kidney but undetectable in any cells of normal adult kidney. These observations seem to imply the involvement of SSRP gene, which is believed to recognize structural alterations of DNA, in kidney development and carcinogenesis of certain types of kidney cancer.     

Adult specific expression and induction of cytochrome P450lpr in house flies

Arabinogalactan-proteins (AGPs) were isolated from the pistils of Nicotiana alata, deglycosylated, and the protein backbones fractionated by reversed-phase HPLC as previously reported. A major fraction, RT35 was isolated and peptide sequences were obtained after protease digestion. A gene-specific degenerate oligonucleotide was designed according to the amino acid sequences and a 380 bp PCR fragment was amplified in vitro from pistil RNA. The PCR fragment was used to screen a pistil cDNA library and a 762 bp cDNA clone (AGPNa3) was isolated and sequenced. The AGPNa3 cDNA encodes a 169 amino acid protein which consists of three domains: an N-terminal secretion signal, a Pro-rich domain and a C-terminal Cys-rich domain. The mature protein has 145 amino acid residues (16.7 kDa) and a predicted pl of 7.5. Northern blot analyses showed that the AGPNa3 gene was only expressed in the pistils of N. alata and of closely related Nicotiana species but not in other plants or suspension-cultured cells. Further Northern blot analysis and in situ hybridization showed that within the pistil, it was primarily expressed in the stigmatic tissues of mature flowers.     

Analysis of a human cDNA containing a tissue-specific alternatively spliced LIM domain

A unique clone, isolated from a human pancreatic cDNA library, was sequenced and characterized. Northern blot analysis showed that the gene is active in a number of fetal and adult tissues, and immunoblots showed expression in nuclear and cytosolic cell fractions. The gene corresponding to the clone was localized to chromosome 13 by human/rodent somatic cell hybrid panels. The largest open reading frame contains a LIM domain, and the deduced peptide from the open reading frame appears to have the characteristics of a LIM-only protein, designated LMO7. RT-PCR and genomic sequence analyses indicate that expression of this gene product is subject to tissue-specific modulation by elimination of the LIM domain by alternative splicing in neural tissues.     

Identification of a breast cancer-specific gene, BCSG1, by direct differential cDNA sequencing

A high-throughput direct-differential cDNA sequencing approach was employed to identify genes differentially expressed in normal breast as compared with breast cancer. Approximately 6000 expressed sequence tags (ESTs) from cDNA libraries of normal breast and breast carcinoma were selected randomly and subjected to EST-sequencing analysis. The relative expression levels of more than 2000 unique EST groups were quantitatively compared in normal versus cancerous breast. Of many putative differentially expressed genes, a breast cancer-specific gene, BCSGC1, which was expressed in high abundance in a breast cancer cDNA library but scarcely in a normal breast cDNA library, was identified as a putative breast cancer marker. In situ hybridization analysis demonstrated stage-specific BCSG1 expression as follows: BCSG1 was undetectable in normal or benign breast lesions, showed partial expression in ductal carcinoma in situ, but was expressed at an extremely high level in advanced infiltrating breast cancer. The predicted amino acid sequence of BCSG1 gene has a significant sequence homology to the non-amyloid beta protein fragment of the Alzheimer's disease amyloid protein. BCSG1 overexpression may indicate breast cancer malignant progression from benign breast or in situ carcinoma to the highly infiltrating carcinoma.     

MR imaging of soft-tissue changes after percutaneous transluminal angioplasty and stent placement

A cDNA library enriched for human fetal-specific liver genes was constructed by suppressive subtractive hybridization. EST fls223 generated from this library was found to represent a novel putative serine/threonine (Ser/Thr) kinase. A full-length clone isolated for this gene encodes a protein of 396 amino acids. The amino acid sequence has 40% identity over 305 amino acids with the B1R Ser/Thr protein kinase of vaccinia virus. This gene has therefore been named VRK1 (vaccinia virus B1R kinase related kinase). VRK1 was also found to have sequence identity (62.0% over 481 nucleotides) to a database EST. A full-length clone for this EST was isolated and sequenced. Conceptual translation predicts a protein of 508 amino acids that, like VRK1, has similarity to B1R kinase (38.7% identity over 300 amino acids). This gene has been named VRK2. Comparison of VRK1 with VRK2 indicates that they encode structurally related putative Ser/Thr protein kinases. Northern analysis shows that expression of both genes is widespread and elevated in highly proliferative cells, such as testis, thymus, and fetal liver. B1R kinase is reported to be essential for DNA replication of vaccinia virus. The similarity of VRK1 and VRK2 to B1R indicates that these genes may have similar functions.     

PA26, a novel target of the p53 tumor suppressor and member of the GADD family of DNA damage and growth arrest inducible genes

We report the cystathionine-beta synthase (CBS) gene expression pattern during early human embryogenesis (3 to 6 weeks post conception) by in situ hybridization and in fetal and adult tissue by Northern Blot analysis. Probes were chosen to recognize either the common sequence to all known CBS mRNAs or the sequences of two different major exons 1 issued of we have previously identified. We demonstrate by in situ hybridization that CBS is continuously expressed from the earliest stages studied (22 days post conception) during embryogenesis in the tissues of developing embryos which will after birth present clinical abnormalities in homocystinuria patients. It is expressed at an especially high level in the neural and cardiac systems until the liver primordium appears. In embryonic central nervous system, the whole neural tube and primary brain vesicles are labeled. Secondary brain vesicles labeling are dependent on the neuroepithelium differentiation. The ventricular layer of the rhombencephalon, cranial nerve nuclei and then after cerebellar cortex derived from rhombencephalon ventricular layer are strongly labeled. Thalamus and other derivatives of the diencephalon plate, the neuroblastic layer of the retina, lens and dorsal root ganglia are labeled. After 35 days post conception, CBS mRNAs was detected in endocardial cells and in cells derived from the neural crest of the heart and in particular developing mesodermic regions such as the primitive hepatocytes of the liver, mesonephros vesicles, various endocrine glands and developing bones. We could not detect tissue specificity of different probes at this embryonic stage. Northern blot analysis consistently detected mRNA species in fetal 25 weeks post conception brain, liver and kidney. The common cDNA probe revealed the 2.5 and 3.7 kb mRNA species from brain, liver and kidney. The exon 1b probe detected only the 2.5 kb mRNA and the exon 1c probe the 3.7 kb mRNA in these three tissues. In adult tissue, the 1b probe detected only the 2.5 kb mRNA and the 1c probe only the 3.7 kb mRNA in the liver.     

Craf-1 protein kinase is essential for mouse development

The complete cDNA of the mouse integral membrane protein 2B gene (Itm2b) was determined by sequence analysis of expressed sequence tag (EST) clone L26775 and a clone isolated from a cDNA library of the osteogenic stromal cell line MN7 (Mathieu et al., 1992. Calcif. Tissue Int. 50, 362-371) and by 5' rapid amplification of cDNA ends (RACE). Alignment of different mouse ESTs confirmed the entire sequence. Northern blot analysis of different neonatal and adult mouse tissues showed that Itm2b is ubiquitously expressed. There are three mRNAs with different lengths in neonatal as well as in adult tissues, originating from alternative polyadenylation by usage of one consensus and two additional variant polyadenylation signals. The cDNA sequence of the human Itm2b homolog (ITM2B) was assembled using data from available human ESTs. Both the mouse and the human gene code for a protein of 266 amino acids (aa) that is homologous to a previously described integral membrane protein, Itm2A, of which the expression is restricted to osteo- and chondrogenic tissues. Itm2A and Itm2B belong to a family of type II integral membrane proteins, which contains a third member, Itm2C (Deleersnijder et al., 1996. J. Biol. Chem. 271, 19475-19482). The human ITM2B and mouse Itm2b genes were previously mapped as unknown ESTs to conserved syntenic regions Homo sapiens 13q12-13 and Mus musculus 14.