Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Alternatively spliced mRNAs encoding the human intraacrosomal protein SP-10 were sought by the reverse transcriptase polymerase chain reaction (RTPCR). Eleven RTPCR products were identified, characterized, and found to represent authentic alternatively spliced SP-10 mRNAs. The 11 alternatively spliced SP-10 mRNAs encoded proteins ranging from 81 to 265 amino acids. The 10 smaller variants all resulted from one or two in-frame deletions in exons 2 and/or 3 of the SP-10 genomic sequence. Quantitative competitive RTPCR showed that the four largest SP-10 mRNAs represented the majority (> 99%) of the SP-10 message in testes from each of four men. The relative abundance of each of the four SP-10 mRNAs varied between individuals, but the longest SP-10 mRNA, SP10-1, which encoded a 265 amino acid protein, was consistently the most abundant, comprising 53-72% of the total SP-10 message. This was followed by the second largest SP-10 mRNA, SP10-2, which encoded a protein of 246 amino acids and comprised 15-32%. The third and fourth largest SP-10 mRNAs, SP10-3 and SP10-4, encoded proteins of 210 and 195 amino acids and accounted for 3.4-8.3% and 8.7-12.5% of the total SP-10 messages, respectively. The remaining 7 SP-10 mRNAs combined accounted for < 1% of the total SP-10 message. Within the low abundance group of mRNAs were two that deleted the entire third exon of SP-10. The present study suggests that phenomena of cryptic splicing and exon skipping occur within the SP-10 mRNA. Along with proteolysis, alternative splicing also helps to explain the heterogeneous forms of SP-10 that have been observed on Western blots of human sperm extracts.
Mol Reprod Dev 1995 May
PMID:Characterization of alternatively spliced human SP-10 mRNAs. 761 99

Large alternatively spliced internal exons are uncommon in vertebrate genes, and the mechanisms governing their usage are unknown. In this report, we examined alternative splicing of a 1-kb internal exon from the human caldesmon gene containing two regulated 5' splice sites that are 687 nucleotides apart. In cell lines normally splicing caldesmon RNA via utilization of the exon-internal 5' splice site, inclusion of the differential exon required a long purine-rich sequence located between the two competing 5' splice sites. This element consisted of four identical 32-nucleotide purine-rich repeats that resemble exon-splicing enhancers (ESE) identified in other genes. One 32-nucleotide repeat supported exon inclusion, repressed usage of the terminal 5' splice site, and functioned in a heterologous exon dependent on exon enhancers for inclusion, indicating that the caldesmon purine-rich sequence can be classified as an ESE. The ESE was required for utilization of the internal 5' splice site only in the presence of the competing 5' splice site and had no effect when placed downstream of the terminal 5' splice site. In the absence of the internal 5' splice site, the ESE activated a normally silent cryptic 5' splice site near the natural internal 5' splice site, indicating that the ESE stimulates upstream 5' splice site selection. We propose that the caldesmon ESE functions to regulate competition between two 5' splice sites within a differential internal exon.
Mol Cell Biol 1995 Aug
PMID:A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon. 762 94

Tropomyosins (TMs) comprise a family of actin-binding proteins which play an important role in the regulation of contractility in muscle (cardiac, skeletal, and smooth) and nonmuscle cells. Although they are present in all cells, different isoforms are characteristic of specific cell types. In vertebrates, there are four different TM genes (alpha-TM, beta-TM, TM30, and TM4), three of which generate alternatively spliced isoforms. This study defines the expression patterns of these isoforms during murine embryogenesis, using both in vivo and in vitro conditions. The embryonic stem cell culture system, which has been shown to mimic different stages of mouse embryonic development, including the differentiation of primitive organ systems such as the myocardium, is used for our in vitro analysis. Our results demonstrate that several TM isoforms are expressed in specific developmental patterns, often correlated with the differentiation of particular tissues or organs. Surprisingly, other TMs, such as the striated muscle beta-TM and smooth muscle alpha-TM, are expressed constitutively. This study also demonstrates that there is an excellent correlation between the expression patterns of the TM isoforms observed in developing embryonic stem cells and mouse embryos. In addition, a quantitative molecular analysis of TM isoforms was conducted in embryonic, neonatal, and adult cardiac tissue. Our results show for the first time that the alpha- and beta-TM striated muscle transcripts are present in the earliest functional stages of the heart, and these TM isoforms are identical to those present throughout cardiac development.
Mol Cell Biol 1993 Jun
PMID:Developmental analysis of tropomyosin gene expression in embryonic stem cells and mouse embryos. 768 95

CFTR mRNA transcripts were analyzed from freshly isolated nasal epithelial cells and lymphocytes (six individuals) and from lymphocytes alone from 14 further individuals. In four of these 20 individuals alternative splicing was observed within the region coding for the first nucleotide binding fold. The RNA sequence between exons 10 and 13 was converted to cDNA and amplified by the polymerase chain reaction (PCR). We detected two PCR products of 583 bp and 464 bp in length. Direct sequencing of both fragments showed that the 583 bp PCR fragment contained an additional 119 bp sequence between exon 10 and exon 11, directly at the normal junction. This insertion contains an in frame stop codon and would, if translated, cause a shift in the reading frame. This stop codon does not result in an undetectable mRNA level as seen with other nonsense mutations within the same region of the CFTR gene (1, 2, own unpublished results). The alternatively spliced mRNA was found to be transcribed from both CF and normal alleles. The 119 bp fragment was amplified from genomic DNA and from the genomic phage TE24V, which includes exon 9, intron 9, exon 10 and a part of intron 10 (3) by PCR using primers created from within the inserted sequence. In addition, the insertion was mapped to a 1Kb EcoRI fragment of phage TE24V by Southern-blot analysis. By sequencing the insert surroundings within the phage TE24V we identified consensus splice sites (donor and acceptor sites, branch point). Furthermore no alterations were detected in the splice site sequences between individuals who express the aberrantly spliced product and those who do not.
Hum Mol Genet 1993 Mar
PMID:Alternative splicing in the first nucleotide binding fold of CFTR. 768 42

We have analyzed the CFTR mRNA populations in a cystic fibrosis patient heterozygous for the 621 + 1G-->T and 711 + 1G-->T mutations. Total RNA isolated from the nasal epithelial cells and Epstein-Barr virus-transformed lymphoblasts derived from this patient was reversely transcribed and a region extending from exon 3 to exon 7 of the gene was amplified by the polymerase chain reaction and analyzed. Three abnormal products were identified, suggesting the presence of three aberrant transcripts, and their profiles were identical in both cell types. Two of the products were found to be missing either exon 4 or exon 5 as anticipated from the transcripts from the 621 + 1G-->T or 711 + 1G-->T alleles, respectively. The third product was apparently derived from an alternatively spliced mRNA species in the absence of the nominal splice site (in 621 + 1G-->T) through the use of a cryptic splice donor sequence (TT528/GTGAGG) within exon 4. Although reading frames appeared to be preserved in all three putative transcripts, significant portions of the presumed first and second transmembrane spans as well as the immediately following cytoplasmic domain would be deleted from the mutant CFTR polypeptides, if made. These observations are consistent with a loss of CFTR function in this cystic fibrosis patient.
Hum Mol Genet 1993 Jun
PMID:Analysis of CFTR transcripts in nasal epithelial cells and lymphoblasts of a cystic fibrosis patient with 621 + 1G-->T and 711 + 1G-->T mutations. 768 8

Glutamate-mediated neurotransmission occurs through the activation of multimeric postsynaptic receptors. One mechanism by which functional diversity of glutamate responsiveness may occur is by a single cell expressing multiple receptors containing different subunits. In a direct test of this hypothesis, we examined the glutamate receptor subunit mRNA composition of several individual CA1 neurons in hippocampal slices. Experiments used amplified antisense RNA coupled with expression profiling and polymerase chain reaction amplification to identify and determine the relative amounts of subunit mRNAs co-localized in single cells. The results demonstrate that each CA1 neuron contains varying amounts of most glutamate receptor mRNAs. In addition to relative mRNA levels, the single-cell approach also highlighted other possible sources of receptor diversity. This included the existence of novel, alternatively spliced forms of the N-methyl-D-aspartate receptor type 1 and glutamate-kainate receptor type 2 subunits. Surprisingly, levels of N-methyl-D-aspartate receptor type 1 mRNA were relatively low, compared with those of other glutamate receptor mRNAs. One postulated source of potential heterogeneity, RNA editing, was not a general cellular mechanism. There was no evidence that glutamate receptor type 5 mRNA was edited in any of the cells that were examined. These data show that individual CA1 neurons, in the intact synaptic network of hippocampal slices, generate glutamate receptor mRNA diversity in several ways, which together contribute to the diversity of functional receptors observed electrophysiologically.
Mol Pharmacol 1993 Aug
PMID:Diversity of glutamate receptor subunit mRNA expression within live hippocampal CA1 neurons. 768 43

Plasma membrane calcium pumps (PMCAs) play a major role in the maintenance and fine regulation of the intracellular Ca2+ concentration. Fourteen subregions of the normal human brain were carefully dissected and analyzed by reverse transcriptase-polymerase chain reaction for the distribution of mRNAs corresponding to the four known PMCA genes as well as their alternative splicing products at two previously defined 'hotspots' A and C. All PMCA genes were found to be expressed in every brain subregion; however, consistent differences were found in the distribution of alternative splice options. The four cortical regions and hippocampus were characterized by the relative preference of variants that include an entire exon at site C and lead to the expression of isoforms of the a-type. Inferior olive and olfactory bulb showed a relative preponderance of the b-form 'default' types of alternative splicing at site C, and a decrease or even the lack of 'differentiated' forms such as variants 1a and 1c. At the N-terminal splice site A, the default x-type variants were predominant in all brain regions for PMCA 1, 3, and 4. By contrast, the pattern of PMCA2 variants was the most variable, ranging from the presence of the entire set of 2x, 2w, and 2z forms in inferior olive to the almost exclusive presence of form 2z (excluding all alternatively spliced sequences) in the four cortical regions, caudate, and hippocampus. Regional differences in the PMCA splice type distribution in normal human brain may correlate with different demands on the regulation of the set-point resting Ca2+ levels in these areas. Changes in these patterns may correlate with altered physiological states of the affected regions and/or reflect an (early) sign of Ca2+ dyshomeostasis characteristic of many neurodegenerative diseases.
Brain Res Mol Brain Res 1995 Feb
PMID:Transcript distribution of plasma membrane Ca2+ pump isoforms and splice variants in the human brain. 772 25

Cyclin E was first identified by screening human cDNA libraries for genes that would complement G1 cyclin mutations in Saccharomyces cerevisiae and has subsequently been found to have specific biochemical and physiological properties that are consistent with it performing a G1 function in mammalian cells. Most significantly, the cyclin E-Cdk2 complex is maximally active at the G1/S transition, and overexpression of cyclin E decreases the time it takes the cell to complete G1 and enter S phase. We have now found that mammalian cells express two forms of cyclin E protein which differ from each other by the presence or absence of a 15-amino-acid amino-terminal domain. These proteins are encoded by alternatively spliced mRNAs and are localized to the nucleus during late G1 and early S phase. Fibroblasts engineered to constitutively overexpress either form of cyclin E showed elevated cyclin E-dependent kinase activity and a shortened G1 phase of the cell cycle. The overexpressed cyclin E protein was detected in the nucleus during all cell cycle phases, including G0. Although the cyclin E protein could be overexpressed in quiescent cells, the cyclin E-Cdk2 complex was inactive. It was not activated until 6 to 8 h after readdition of serum, 4 h earlier than the endogenous cyclin E-Cdk2. This premature activation of cyclin E-Cdk2 was consistent with the extent of G1 shortening caused by cyclin E overexpression. Microinjection of affinity-purified anti-cyclin E antibodies during G1 inhibited entry into S phase, whereas microinjection performed near the G1/S transition was ineffective. These results demonstrate that cyclin E is necessary for entry into S phase. Moreover, we found that cyclin E, in contrast to cyclin D1, was required for the G1/S transition even in cells lacking retinoblastoma protein function. Therefore, cyclins E and D1 control two different transitions within the human cell cycle.
Mol Cell Biol 1995 May
PMID:Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. 773 42

Multiple alternatively spliced 5' untranslated regions (5'UTRs) have been identified in growth hormone (GH) receptor mRNA isolated from hepatic and adipocyte tissue. In the present study, the preferential utilisation of a GC-rich 5'UTR, designated exon 1B, was observed following the isolation of ovine (o) GH receptor cDNA clones from a skeletal muscle cDNA library. Although exon 1B-oGH receptor mRNA was expressed in all tissues examined, marked differences in the level of expression relative to the whole GH receptor transcript pool were observed between tissues. A single genomic clone (lambda 9) was isolated that encompassed exon 1B, together with 6 kilobase pairs of 5' and 12 kilobase pairs of 3' flanking sequence. Multiple transcription initiation sites were identified using RNase protection analysis on skeletal muscle poly(A)+ RNA, a result consistent with the absence of a proximal TATA box element. A CAAT box (-37 to -33) and a putative binding site for SP1 (a GC box -68 to -63) were found in the sense orientation immediately upstream of major transcription initiation site. Transfection of a series of overlapping promoter fragments linked to the luciferase reporter gene into HuH7, CHO and HeLa cells defined a core promoter element of 134 base pairs that was sufficient for maximum promoter activity. The emerging complexity of the 5' regulatory region of the GH receptor gene was emphasised by the observation that probes derived from exon 1B and the distal 3' intron boundary do not hybridise with previously cloned genomic sequences that span the liver-specific P1 promoter and exon 2.
Mol Cell Endocrinol 1995 Feb 27
PMID:Differential expression of growth hormone receptor messenger RNA from a second promoter. 775 37

PRL has been shown to induce a number of genes after the stimulation of quiescent Nb2 T-cells, including c-fos, c-myc, ornithine decarboxylase, interferon regulatory factor-1, and others. One of these genes, LHRH, has not previously been reported to respond in this manner, although we and others have reported its presence in rat and human T- and B-cells. Furthermore, recent evidence suggests that LHRH functions as an immunoregulator in a cytokine-like manner. Using the rat immature T-cell line Nb2, we present data showing for the first time that 1) the LHRH gene is regulated by PRL at various times during the cell cycle; 2) an alternatively spliced LHRH messenger RNA exists in Nb2 cells and may produce a new truncated GnRH-associated peptide (alternatively called PIF for PRL-inhibiting factor); 3) the LHRH receptor is expressed in lymphocytes in a manner similar to the LHRH gene after PRL addition, and its complementary DNA sequence is identical to that of the pituitary receptor; 5) the SH gene, found on the opposite strand of the LHRH gene, is expressed in lymphocytes at the same time and in the same manner as the LHRH gene; 6) the LHRH messenger RNA has a very short half-life in these cells; and 7) the lymphocyte LHRH transcription start site is essentially the same as the hypothalamic site. These data strengthen the relationship between PRL and LHRH expression in the immune system and further support our contention that LHRH is an important immunoregulator, on par with other known cytokines.
Mol Endocrinol 1995 Jan
PMID:Coordinate gene expression of luteinizing hormone-releasing hormone (LHRH) and the LHRH-receptor after prolactin stimulation in the rat Nb2 T-cell line: implications for a role in immunomodulation and cell cycle gene expression. 776 Aug 50


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