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PubMed1991

DNase activity in Costa Rican crotaline snake venoms: quantification of activity and identification of electrophoretic variants.

DNase activity of Costa Rican crotaline snake venoms from the genera Bothrops, Crotalus and Lachesis was quantified by an enzymodiffusion method on agarose/DNA gels containing ethidium bromide. The reaction is detected as a ring lacking fluorescence when gels are visualized under u.v. light. Electrophoresis of non-fluorescent areas demonstrated DNA degradation. All of the venoms had DNase activity, B. schlegelii being most active. Venoms from B. schlegelii and B. asper induced an inner hyper-fluorescent ring in addition to the external non-fluorescent ring, probably caused by the formation of complexes between DNA and highly basic proteins present in these venoms. In order to study the number of electrophoretic DNase variants, venoms were separated by analytical isoelectric focusing on polyacrylamide minigels, proteins were transferred to nitrocellulose paper and the paper was placed over an agarose gel containing DNA. Then the agarose gel was stained with ethidium bromide and the bands of DNase activity were visualized under u.v. light. All the venoms tested, as well as commercial DNase showed several bands with DNase activity. The majority of venom DNase variants have basic pIs although bands with acidic pIs were also present in B. godmani and L. muta venoms. No major differences in the DNase electrophoretic pattern were observed between individual venoms of adult B. asper specimens nor between lyophilized and frozen venoms.

Crotalid Venoms↗
PubMed1995

Low-stringency single specific primer PCR, DNA sequencing and single-strand conformation polymorphism of PCR products for identification of genetic variants of human papillomavirus type 16.

Two fragments from within the long control region of the genome of human papillomavirus type 16 (HPV16) were amplified using the polymerase chain reaction (PCR). Putative genetic variation among the parent viruses was assessed by complete sequence analysis and single-strand conformation polymorphism (SSCP) analysis of one of the fragments and by application of the recently described low-stringency single specific primer (LSSP) PCR to both PCR products. The study comprised 34 HPV16 positive samples, derived from seventeen different individuals. It is demonstrated that under experimentally standardised conditions the LSSP PCR-, sequencing- and SSCP-data display differing degrees of resolution. Based on combined LSSP PCR analyses, 33 out of 34 samples can be discriminated, whereas SSCP and direct sequencing identify 4 and 8 types, respectively. Although the variability observed among LSSP PCR patterns may be the consequence of small quantities of mutated viral amplimers, no concordant grouping of strains, identical by sequencing and SSCP analysis, can be established by either of the four theoretically possible LSSP PCR assays. Results are discussed in the context of experimental variability of the procedures or genetic heterogeneity of HPV16 pools derived from cervical swabs.

Base Sequence↗
PubMed1995

dlk, pG2 and Pref-1 mRNAs encode similar proteins belonging to the EGF-like superfamily. Identification of polymorphic variants of this RNA.

dlk encodes a transmembrane protein member of the EGF-like family of homeotic proteins. dlk is expressed in the same type of neuroendocrine tissues and tumors as pG2, a gene cloned because of its differential expression in human pheochromocytomas versus neuroblastomas. Human dlk and pG2 cDNAs are around 98% similar in sequence, but the predicted proteins encoded by those genes are apparently unrelated. This fact suggested the existence of polymorphic variants of the same gene. We have sequenced again several pG2 and dlk clones in parallel. We identified a pG2 cDNA species corresponding to an alternatively spliced dlk mRNA, as well as several other variant forms of dlk mRNA. One of the pG2 clones resulted to be identical to human dlk and encode the same EGF-like protein. Pref-1, a cDNA isolated from 3T3-L1 fibroblasts, encodes a putative protein possessing an extracellular EGF-like domain similar to dlk, but a different intracellular region. Analysis of sequence data from different clones obtained in our laboratory confirmed some of the differences between dlk and Pref-1. However, the putative difference in the intracellular regions of dlk and Pref-1 was due to sequence artifacts. These data suggest that dlk, pG2 and Pref-1 are variant products of the same gene.

Amino Acid Sequence↗
PubMed1994

Limited polymorphism of the HLA-DQA2 promoter and identification of a variant octamer.

Previous studies have suggested that the HLA-DQA2 gene may be associated with IDDM. The apparently limited allelism at this locus prompted us to investigate whether this association might be with the level of gene expression rather than with specific alleles. The proximal promoter region of HLA-DQA2 was sequenced in three homozygous DR4;DQ8 subjects with IDDM, six homozygous DR3;DQ2 subjects (three healthy controls and three with IDDM), and selected DR4 and DR6 cell lines. This 388-bp region encompassed the known control W/Z/H/S, X, and Y boxes and included a previously unremarked variant octamer sequence 40 bp upstream of the transcription start site. Only one polymorphic site was present among these 15 sequences, found in one DR3;DQ2 subject and a DR6;DQ6 cell line. This indicates that any disease association with HLA-DQA2, at least among DR3;DQ2 individuals, cannot be accounted for solely by polymorphism of the proximal promoter region.

Amino Acid Sequence↗
PubMed2005

Identification of DLK1 variants in pituitary- and neuroendocrine tumors.

In a gene chip analysis of common pituitary tumor types, one of the genes with the most impressive tissue-specific expression regulation was delta-like 1 (DLK1), which was strongly expressed in GH-secreting (GH-S) pituitary tumors. In addition to pituitary adenomas, various endocrine tumors were subjected to real-time-quantitative PCR revealing high expression of DLK1 in normal pituitary tissue, in GH-S-, in one prolactin-secreting pituitary adenoma and in pheochromocytomas. Additionally, three DLK1 gene-derived subvariants were identified. The first, lacking 204 bp--coding for epidermal growth factor-like domain 6 and parts of the juxtamembrane region--was named Secredeltin. In the other two splice variants (named Brevideltin and Brevideltinin), a stop codon is introduced due to a frame-shift, leading to truncated proteins of 204 and 213 aas, respectively.

Alternative Splicing↗
PubMed2004

Identification of allelic variants of the bovine immune regulatory molecule CEACAM1 implies a pathogen-driven evolution.

Carcinoembryonic antigen-related cell adhesion molecule (CEACAM1), the primordial member of the carcinoembryonic antigen (CEA) family, functions as a MHC-independent natural killer (NK) cell inhibitory receptor, regulates T and B cell proliferation, and induces dendritic cell (DC) maturation. Despite these fundamental functions, CEACAM1 and most of the CEA family members differ significantly in primates and rodents. A number of diverse murine and human pathogens use CEACAM1 as a cellular receptor, indicating that the observed species-specific differences are the result of divergent molecular pathogen/host coevolution. To gain deeper insight into its evolution and function, we cloned CEACAM1 cDNA from cattle as a representative of a third mammalian order. Bovine CEACAM1 differs considerably from rodent and primate CEACAM1 due to deletion of the B domain exon which was most likely caused by insertion of LINE/SINE sequences and reveals alternative splicing within the transmembrane exon. However, the characteristic long and short isoforms exist which contain or lack the typical immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their cytoplasmic tails, respectively. Bovine peripheral blood lymphocytes (PBL) express only ITIM-containing CEACAM1 isoforms, and upregulate their expression upon stimulation, suggesting an inhibitory function in these cells. As found in rodents, two clearly distinct CEACAM1 alleles exist in cattle. In the a allele, a unique deletion of three amino acids is found in the N domain, which is important for pathogen binding in mice and humans. This is consistent with the notion that CEACAM1 serves or has served as a pathogen receptor in cattle.

Alleles↗
PubMed2004

Identification of molecular variants at the promoter region of the human alpha 7 neuronal nicotinic acetylcholine receptor subunit gene but lack of association with schizophrenia.

The human alpha7 neuronal nicotinic receptor subunit gene has been considered as a candidate gene for P50 sensory gating deficit in schizophrenic patients. Because P50 sensory gating deficit is a common neurophysiological dysfunction in patients with schizophrenia and schizophrenia spectrum disorders, it is conceivable to hypothesize that the human alpha7 neuronal nicotinic receptor subunit gene might be a susceptible gene for schizophrenia. Researchers have reported that mutations in the protein-coding sequences of the human alpha7 neuronal nicotinic receptor subunit gene are very rare. Therefore, we searched for mutations at the promoter region of the human alpha7 neuronal nicotinic receptor subunit gene and performed a genetic association study in 249 unrelated Han Chinese schizophrenic patients and 273 non-psychotic subjects from Taiwan. Two molecular variants were identified and designated g.-213G>A and g.-324A>G, respectively. The g.-213G>A variant was found to obliterate a putative NF-1 transcription factor binding site using computer analysis. One out of 249 patients was detected to be a heterozygote for this variant, but none of 273 control subjects was. The g.-324A>G variant was also very rare in both patients and control subjects, only one heterozygote of this variant was identified in 249 patients and 273 control subjects, respectively. Hence, in this study, we did not find mutations in the human alpha7 neuronal nicotinic receptor subunit gene that are associated with schizophrenia in our population.

Adult↗
PubMed2003

Identification of a variant form of PZR lacking immunoreceptor tyrosine-based inhibitory motifs.

PZR is an immunoglobulin superfamily protein that specifically binds tyrosine phosphatase SHP-2 through its intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Here we report a novel isoform of the protein designated PZR1b. PZR1b shares the same extracellular region with PZR, but it lacks intracellular ITIMs and thus the ability to recruit SHP-2. Genomic sequence analysis revealed that PZR1b is resulted from alternative gene splicing of the PZR gene localized at chromosome 1q24. Like PZR, PZR1b is widely expressed. However, the relative ratio of two forms varies in different human tissues and cells. More importantly, overexpression of PZR1b in human HT-1080 cells had a dominant negative effect by blocking concanavalin A-induced tyrosine phosphorylation of full-length PZR and recruitment of tyrosine phosphatase SHP-2. Therefore, PZR1b may have an important role in cell signaling by counteracting with PZR.

Alternative Splicing↗
PubMed1998

Identification of sequence variants and analysis of the role of the catechol-O-methyl-transferase gene in schizophrenia susceptibility.

BACKGROUND: Deletions of 1.5-2 MB of chromosome 22q11 have been previously associated with schizophrenia. The deleted region includes proximally the region harboring genes involved in DiGeorge and velocardiofacial syndromes. Distally, it includes the gene for catechol-O-methyl-transferase (COMT), an enzyme that catalyzes the O-methylation of catecholamine neurotransmitters, including dopamine, and which therefore is considered a candidate gene for schizophrenia. METHODS: We address the issue of a direct involvement of the COMT gene in the development of schizophrenia by employing the first extensive mutational analysis of this gene in a sample of 157 schizophrenia patients and 129 healthy controls, using single-strand conformation polymorphism and chemical cleavage methodologies. RESULTS: No mutations were found, but several sequence variants were identified, including the genetic polymorphism that underlies the high/low activity of the enzyme (a Val158-->Met change, which results in the creation of an NlaIII restriction site in the low-activity allele). The distribution of the NlaIII genotypes among subsets of schizophrenia patients was analyzed. CONCLUSIONS: The results presented here argue against a major role of COMT in schizophrenia in general (although a minor effect could not be excluded) and represent a first step toward a more refined delineation of the phenotype/genotype relationship between 22q11 microdeletions and schizophrenia susceptibility.

Adult↗
PubMed1988

Quantification of hemoglobin A2 and identification of hemoglobin variants using a fully automated hemoglobin analyzer.

The DIAMAT Analyser System is a fully automated high performance liquid chromatographic (HPLC) instrument originally designed for the quantification of glycated hemoglobin (HbA1c). Buffers were developed for the separation and quantification of hemoglobin A2 on the DIAMAT. Also studied were the retention patterns of various hemoglobin variants on the DIAMAT using the buffers developed for the hemoglobin A2 quantification.

Adult↗
PubMed1998

Identification of allelic variants in the follicle-stimulating hormone receptor genes of females with or without hypergonadotropic amenorrhea.

OBJECTIVE: To determine whether women with idiopathic hypergonadotropic amenorrhea have unique alterations in the FSH receptor gene that could account for reduced activity. DESIGN: Compare FSH receptor genes of affected women with normally menstruating control subjects. SETTING: Center for Reproductive Health and university departments. PATIENT(S): Fourteen female subjects, including four normally menstruating controls; four sibling sisters, two of whom developed premature ovarian failure (POF); four patients with POF; one patient with 46,XX gonadal dysgenesis (GD); and one patient with hypogonadotropic hypogonadism. INTERVENTION(S): Blood samples were collected. MAIN OUTCOME MEASURE(S): Restriction fragment length polymorphism (RFLP) analysis, single-stranded conformation polymorphism analysis, gel electrophoretic mobility of amplified genomic DNA, and FSH receptor gene sequence. RESULT(S): The DNA sequencing revealed allelic variants in one RFLP-positive control. There were two silent variants and one missense variant that resulted in a change from Asp to Gly at position 334 from the start Met in the amino acid sequence. Six of 10 subjects, including controls and patients with POF and GD, had an allelic variant in which A was changed to G at position 919 which caused Thr307 to be changed to Ala. CONCLUSION(S): Allelic variants in the FSH receptor gene occur commonly in control subjects and affected patients.

Alleles↗
PubMed2001

Characterization of the mouse integrin subunit alpha10 gene and comparison with its human homologue. Genomic structure, chromosomal localization and identification of splice variants.

Alpha10beta1 is a collagen-binding integrin expressed by chondrocytes [Camper et al. (1998) J. Biol. Chem. 273: 20383-20389]. In the present study, the mouse alpha10 gene was isolated from a sCos1 SVJ library and the genomic structure and chromosomal localization was determined. The alpha10 gene consists of 30 translated exons spanning a region of approximately 18 kb genomic DNA. The sequences of all exon/intron borders follow the consensus "gt-ag" rule. A transcription start site, determined by primer extension analysis, was located 38 nucleotides upstream of the initiation ATG site. The 5' flanking region of the transcription start site lacked a TATA-box. The first exon contained, in addition to 38 untranslated nucleotides, the ATG translation start site and the major part of the signal peptide. The alpha10 gene was mapped to mouse chromosome 3 by fluorescence in situ hybridization and is the only integrin subunit localized to this chromosome. When we investigated the expression of alpha10 by PCR we found that both mouse and human articular chondrocytes express extracellular splice variants of the alpha10 subunit. In mouse, exon 26 was extended into the intron by 62 nt, generating a truncated alpha10-chain. In human, exon 25 consisted of 114 nt which were alternately spliced in or out.

Alternative Splicing↗
PubMed2001

Identification of genetic variants in the human thromboxane synthase gene (CYP5A1).

Thromboxane synthase (CYP5A1) catalyzes the conversion of prostaglandin H2 to thromboxane A2, a potent mediator of platelet aggregation, vasoconstriction and bronchoconstriction. It has been implicated in the patho-physiological process of a variety of diseases, such as atherosclerosis, myocardial infarction, stroke and asthma. On the basis of the hypothesis that variations of the CYP5A1 gene may play an important role in human diseases, we performed a screening for variations in the human CYP5A1 gene sequence. We examined genomic DNA from 200 individuals, for mutations in the promoter region, the protein encoding sequences and the 3'-untranslated region of the CYP5A1. Eleven polymorphisms have been identified in the CYP5A1 gene including eight missense mutations R61H, D161E, N246S, L357V, Q417E, E450K, T451N and R466Q. This is the first report of genetic variants in the human CYP5A1 altering the protein sequence. The effect of these variants on the metabolic activity of CYP5A1 remains to be further evaluated.

Alleles↗