The mouse adducin gene family: alternative splicing and chromosomal localization

Mouse cDNA sequences encoding α, β, and γ adducins were cloned from a mouse reticulocyte cDNA library. The purified clones contain alternatively spliced exons from all three adducin genes. In the case of α and β, the inclusion of the alternatively spliced exons results in truncated polypeptide isoforms (called α-2 and β-2). The mouse predicted amino acid sequences are compared with published rat and human sequences. For completion of this comparison, cDNA encoding the rat β-1 carboxy terminus was cloned by PCR. The carboxy terminal region containing MARCKS homology, calmodulin-binding region-2, and spectrin-actin-binding site, is conserved among α-1, β-1, and γ-1 isoforms in mouse, rat, and humans. We also report here the localization of the gene encoding γ adducin (Add3) to murine Chr 19, in a region that shows conserved synteny with human Chr 10. Received: 1 June 1999 / Accepted: 25 August 1999     

A novel human cytochrome P450 gene (P450IIB): chromosomal localization and evidence for alternative splicing.

We have isolated from a single human liver cDNA library two clones which are highly homologous (78% over the coding region) to the major phenobarbital-inducible P450 from rat (P450IIB1). This is the first direct demonstration of the presence of the P450IIB gene subfamily in humans. This subfamily is much less extensive than the rodent homologues, but does appear to contain at least two genes. Of the cDNA clones isolated one is apparently normally spliced, whereas the other lacks exon 8 and retains all or part of intron 5. Both clones contain transcribed Alu sequences. The human P450IIB gene has been located to chromosome 19q12----19q13.2 using a probe derived from intron 5, and is close to the CYP 2A locus encoding cytochrome P450IIA2. Restriction fragment length polymorphisms have been found with the enzymes BamHI and MspI which will enable linkage to be determined between these two loci.     

Resolving deconvolution ambiguity in gene alternative splicing

Background  

For many gene structures it is impossible to resolve intensity data uniquely to establish abundances of splice variants. This was empirically noted by Wang et al. in which it was called a "degeneracy problem". The ambiguity results from an ill-posed problem where additional information is needed in order to obtain an unique answer in splice variant deconvolution.     

The dopamine D2 receptor: two molecular forms generated by alternative splicing.

Cloned human dopamine D2 receptor cDNA was isolated from a pituitary cDNA library and found to encode an additional 29 amino acid residues in the predicted intracellular domain between transmembrane regions 5 and 6 relative to a previously described rat brain D2 receptor. Results from polymerase chain reactions as well as in situ hybridization revealed that mRNA encoding both receptor forms is present in pituitary and brain of both rat and man. The larger form was predominant in these tissues and, as shown in the rat, expressed by dopaminergic and dopaminoceptive neurons. Analysis of the human gene showed that the additional peptide sequence is encoded by a separate exon. Hence, the two receptor forms are generated by differential splicing possibly to permit coupling to different G proteins. Both receptors expressed in cultured mammalian cells bind [3H]spiperone with high affinity and inhibit adenylyl cyclase, as expected of the D2 receptor subtype.     

Immunological evidence for a P2 protamine precursor in mature rat sperm.

High molecular weight proteins in Rattus norvegicus that are immunoreactive with an anti-protamine 2 specific antibody but not with an anti-protamine 1 specific antibody are described. These proteins were detected by coupling high-performance liquid chromatography (HPLC) with an enzyme-linked immunosorbent assay (ELISA). Briefly, following HPLC separation of rat sperm nuclear proteins, the HPLC fractions were probed with the antibodies. We estimate that the antibody probes are 100-1000 times more sensitive than UV absorbance measurements. Immunoblot analysis following acid-urea electrophoretic separation of rat sperm nuclear proteins, and of the HPLC fractions, also detected putative protamine 2 precursor proteins. The proteins reactive with the anti-protamine 2 antibody are most likely not mature protamine 2, since they were detected in a region of the chromatogram where we would not expect protamine 2 to migrate based on the chromatographic locations of human and mouse protamine 2. Likewise, the immunoblotting experiments demonstrated that the anti-protamine 2 antibody recognized proteins with slower electrophoretic mobilities than would be expected for a mature protamine 2. An anti-protamine 1 monoclonal antibody, Hup1N, that binds rat protamine 1 is also described. Hup1N allowed for identification of the HPLC fractions that contained rat protamine 1. Finally, we demonstrated that Hup1N binds protamine 1 from a large number of species, suggesting a conserved epitope for Hup1N.