Structure and alternative splicing of the presenilin-2 gene.

Missense mutations in the presenilin-1 (PS-1) and presenilin-2 (PS-2) genes have been shown to be causes of autosomal dominant Alzheimer's disease (the AD3 and AD4 loci, respectively). Alternative splicing has previously been reported in the PS-1 gene. In this study, elucidation of intron/exon boundary sequences revealed that PS-2 is encoded by 10 coding exons. In addition, PS-2 cDNA cloning and RT-PCR using RNA from a variety of normal tissues revealed the presence of alternatively spliced products. These products included species with in frame omissions of exon 8 and simultaneous omissions of exons 3 and 4.

Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence.

In an effort to identify new genes and analyse their expression patterns, 174,472 partial complementary DNA sequences (expressed sequence tags (ESTs)), totalling more than 52 million nucleotides of human DNA sequence, have been generated from 300 cDNA libraries constructed from 37 distinct organs and tissues. These ESTs have been combined with an additional 118,406 ESTs from the database dbEST, for a total of 83 million nucleotides, and treated as a shotgun sequence assembly project. The assembly process yielded 29,599 distinct tentative human consensus (THC) sequences and 58,384 non-overlapping ESTs. Of these 87,983 distinct sequences, 10,214 further characterize previously known genes based on statistically significant similarity to sequences in the available databases; the remainder identify previously unknown genes. Thirty tissues were sampled by over 1,000 ESTs each; only eight genes were matched by ESTs from all 30 tissues, and 227 genes were represented in 20 or more of the tissues sampled with more than 1,000 ESTs. Approximately 40% of identified human genes appear to be associated with basic energy metabolism, cell structure, homeostasis and cell division, 22% with RNA and protein synthesis and processing, and 12% with cell signalling and communication.

Comparative expressed-sequence-tag analysis of differential gene expression profiles in PC-12 cells before and after nerve growth factor treatment.

Nerve growth factor-induced differentiation of adrenal chromaffin PC-12 cells to a neuronal phenotype involves alterations in gene expression and represents a model system to study neuronal differentiation. We have used the expressed-sequence-tag approach to identify approximately 600 differentially expressed mRNAs in untreated and nerve growth factor-treated PC-12 cells that encode proteins with diverse structural and biochemical functions. Many of these mRNAs encode proteins belonging to cellular pathways not previously known to be regulated by nerve growth factor. Comparative expressed-sequence-tag analysis provides a basis for surveying global changes in gene-expression patterns in response to biological signals at an unprecedented scale, is a powerful tool for identifying potential interactions between different cellular pathways, and allows the gene-expression profiles of individual genes belonging to a particular pathway to be followed.

Expression, cytoskeletal utilization and dimer formation of tropomyosin derived from retroviral-mediated cDNA transfer. Metabolism of tropomyosin from transduced cDNA.

Expression of the tropomyosin-1 isoform was enhanced by cDNA transfer in non-transformed murine 3T3 fibroblasts and also in v-Ki-ras transformed fibroblasts in which native tropomyosin-1 expression had been reduced and tropomyosin-2 synthesis virtually eliminated by action of the oncogene. The level of synthesis of insert-derived tropomyosin-1 was similar in normal and transformed transductants (3-5 times normal levels). The high level of insert-derived tropomyosin-1 expression resulted in a considerable increase in tropomyosin-1 utilization in the cytoskeleton of transformed cells, but this expression still did not reach normal levels, suggesting an oncogene-related inhibition of tropomyosin utilization. A large proportion of newly synthesized native tropomyosin-1 in normal, unmodified fibroblasts appeared in homodimers which, upon prolonged incubation, were largely converted to the heterodimers. Excess tropomyosin-1 derived from the inserted cDNA also appeared largely as the homodimer in both normal and transformed cells. This homodimer was utilized effectively in the formation of cytoskeletal structures but was partially converted to heterodimer by chain exchange. Under steady-state conditions, approximately 33% of the cytoskeletal tropomyosin-1-containing dimers were homodimers, compared to approximately 10% in normal fibroblasts. The results show that the increased amount of tropomyosin-1 homodimer entering the cytoskeleton under conditions of tropomyosin-1 excess, results in an atypical microfilament composition. The effect of this excess of tropomyosin-1 homodimers on stability or function of microfilament fibers remains to be determined. The results also confirm that the mechanisms of rapid homodimer formation with conversion to heterodimers by chain exchange, known from in vitro studies, also occur in vivo.

Expression of transduced tropomyosin 1 cDNA suppresses neoplastic growth of cells transformed by the ras oncogene.

Synthesis of certain members of the tropomyosin family of microfilament-associated proteins is suppressed in fibroblasts neoplastically transformed by a number of retroviral oncogenes, by transforming growth factor alpha, and by chemical mutagens. To test whether tropomyosin suppression is a required event in neoplastic transformation, expression of one of two suppressed tropomyosins in NIH 3T3 mouse cells transformed by the ras oncogene was restored by retrovirally mediated cDNA transfer. Cells expressing the inserted cDNA showed partial restoration of microfilament bundle formation (which is typically deranged in transformed cells) together with increased cytoplasmic spreading. More importantly, they lost anchorage-independent growth capability, and the onset of tumor growth in athymic mice was delayed. When tumors arose they no longer expressed the inserted cDNA. These observations support the conclusion that tropomyosin suppression is a necessary event for the expression of components of the transformed phenotype, particularly with respect to anchorage-independent growth and tumorigenesis, which correlate closely with neoplastic potential. This potentially reversible requirement may link different initial events produced by a variety of oncogenic modalities to a common pathway leading to neoplastic growth.

Accumulation and localization of troponin-T in developing hearts of Ambystoma mexicanum.

Troponin-T (Tn-T) expression in developing hearts of axolotls, Ambystoma mexicanum, was studied with the use of polyclonal and monoclonal antibodies and SDS-polyacrylamide gel electrophoresis. In precontractile hearts (stage 32/33), Tn-T was present in addition to myosin, actin and tropomyosin as evidenced by the presence of the protein bands in SDS-gels and by indirect immunofluorescence. Tn-T was localized in amorphous collections at the peripheries of these precontractile cells. Hearts of normal and cardiac lethal mutant siblings were also analysed for Tn-T expression. No detectable differences in the quantity of protein present was observed by gel electrophoresis or by indirect immuno-fluorescence. The most striking difference concerned the localization of the protein. In normal hearts, Tn-T was primarily localized in the I-bands of organized myofibrils; however, in mutant cells the Tn-T was localized in amorphous collections at the cell peripheries suggesting a reduction of myofibrillar organization in these cells. No differences were observed in the contractile protein composition between normal and mutant embryonic hearts by gel electrophoresis experiments.

Immunofluorescence studies for myosin, alpha-actinin and tropomyosin in developing hearts of normal and cardiac lethal mutant Mexican axolotls, Ambystoma mexicanum.

Recessive mutant gene c in axolotl embryos results in an absence of normal heart function. Immunofluorescence studies were done to determine the distributions of myosin, tropomyosin and alpha-actinin in the hearts of normal and mutant siblings. Anti-myosin specifically stains the A bands of myofibrils in normal hearts and reveals a progressive increase in myofibril organization with development. Mutant hearts display less staining for myosin than normal and localization is mainly in amorphous collections. Anti-alpha-actinin stains the Z lines of myofibrils in normal myocytes. Mutant cells also have significant staining for alpha-actinin but show no striations. Antitropomyosin intensely stains the I bands of myofibrils in normal cells; however, there is very little staining for tropomyosin in mutant hearts. Thus, mutant myocardial cells have reduced but significant amounts of actin (Lemanski, Mooseker, Peachey & Iyengar, 1976) and myosin, even though non-filamentous, and substantial amounts of alpha-actinin. The cells appear to contain little tropomyosin.