Alternative Protein Products Research Articles

Structural Organization of the Spliced Immediate-Early Gene Complex that Encodes the Major Acidic Nuclear (IE1) and Transactivator (IE2) Proteins of African Green Monkey Cytomegalovirus.

Total immediate-early (IE) RNA synthesized after infection with African green monkey cytomegalovirus (SCMV) in the presence of cycloheximide contained a major 2.3-kb mRNA species that acted as template for in vitro synthesis of a single 94-kD nuclear protein. The same IE RNA hybridized predominantly to a 1.8-kb subregion of the 220-kb genome which mapped 1.5 kb to the left of the in vitro transcription start site and TATATAA motif previously associated with the powerful MIE (IE94) enhancer region. However, DNA sequence and S1-mapping analysis of a 5-kb region downstream from the promoter revealed the existence of a far upstream noncoding first exon and four additional spliced exons capable of encoding two alternative protein products with shared N-terminal domains. This region is similar in structure to that of the MIE gene complex of human cytomegalovirus (HCMV), including being highly CpG suppressed. Exons 2, 3, and 4 encode an acidic protein equivalent to the 68-kD IE1 protein (UL123) of HCMV and exons 2, 3, and 5 encode a protein equivalent to the 80-kD IE2 (UL122) DNA-binding protein of HCMV. Transcripts from across the IE2 region were detected within the cycloheximide RNA, but they were present at 10- to 20-fold lower abundance than IE1 transcripts. The proposed 547-codon IE1 (IE94) acidic phosphoprotein of SCMV displays minimal residual homology with the IE1 protein of HCMV, but both associate with metaphase chromosomes and have large C-terminal glutamic-acid-rich domains. In contrast, the proposed 583-codon IE2 protein of SCMV displays extensive amino acid similarity to the HCMV IE2 transcriptional regulatory protein especially within C-terminal domains that are known to play a major role in promoter targeting for both transactivation and negative autoregulation functions. Copyright 1995 S. Karger AG, Basel

Rat granulosa cells express the gonadotrophin-releasing hormone gene: evidence from in-situ hybridization histochemistry.

There is still debate as to whether natural sequence gonadotrophin-releasing hormone (GnRH) is produced in the mammalian gonads and concerning its potential role as a paracrine modulator of gonadal function. To address this question, we have used in-situ hybridization histochemistry with an oligonucleotide probe complementary to the GnRH decapeptide coding sequence, to determine the cellular site(s) of expression of the GnRH gene in rodent ovaries. GnRH mRNA was detected in granulosa and thecal cells from ovarian follicles at all stages of development (primary-->Graafian), with no significant change in grain density during follicular development. The granulosa cell compartment always contained more mRNA than the thecal cell compartment. Corpora lutea expressed the GnRH gene to the same extent as thecal cells. These results indicate that preproGnRH mRNA is detectable under physiological conditions in the mammalian ovary, though whether this produces authentic GnRH decapeptide or an alternative protein product is not known. The physiological significance of these findings remains to be determined.

Alternative mRNA splicing: the Shaker gene

*Department of Molecular Biolo~ and tDivision of Preclinical Neuroscience and Endocrinolo~, Research Institute of SC~S Clinic, 106r;6 N. Torrey Pines Road La ]olla, CA 92037, USA. One of the surprising findings from the past decade of molecular biology researchis that a single gene can give rise to more than one mature mRNA via the process of alternative mRNA splicing. This phenomenon occurs in a substantial proportion of genes: for example, of known genes expressed predominantly in the nervous system, some 30% utilize alternative splicing. Most alternative splicing events af- fect the region of the mRNA that encodes protein, thus one biolo~cal rationale for this process is apparent: the alternative mRNAs encode pro- teins that share many characteristics with each other but differ in those properties harbored by the alter- natively ceded regions. Thus, the alternative protein products of a gene represent a protein family. Potassium channels The functional significance of al- ternative splicing is made extremely evident in molecular studies of voltage-gated potassium channels. Action potentials are initiated by depolarizations of the axon mem- brane that activate an inward Na + conductance. Action potentials are self-limiting in that, as membranes become depolarized, the conduc- tance to outward flow of K + also increases, thereby restoring, after 0.2-0.5ms, the membrane potential and the resting ,;alue of the Na + conductance. Several K + channels mediate membrane repolarization and these have been found to vary in their electrophysiological proper- ties, specifically in the rates at which they cause repolarization. Because the duration of depolarization at nerve terminals determines the amount of transmitter released, the type of K + channel expressed at a particular terminal may specify the strength of its synaptic interaction. To understand the variation in the electrophysiological properties of K + channels, it was essential to under- stand the biochemical nature of the different channel types. However, at the time there was neither antibody nor protein sequence information available to use in the construction of molec-l~r probes for cDNA doning. Instead, the characterization of K + channels was initiated by analysis of the Shaker (Sh) gene of Drosophila ~hu~aster and offers an example of how one may proceed from a gene to a description of its products. Sbakerflies Drosophila Sb mutants were initi- ally identified because, after ether anesthesia, the legs of mutant flies shook. The mutants exhibit an al- tered K + current identified as the A- current (Ia). The altered current was associated with prolonged action potentials and increased neurotrans-

Alternative protein products with different carboxyl termini from a single class I gene, H-2Kb.

In addition to mRNA encoding the canonical form of the murine class I antigen H-2Kb (348 amino acid residues), mRNA that would encode a shortened form of H-2Kb (missing 9 amino acids from the C-terminus) has been identified in C57BL/6 spleen cells by RNase-protection studies. The alternative transcripts of H-2Kb arise through the use of different AG acceptor splice sites for exon VIII. The existence of a shortened H-2Kb protein was demonstrated by sequential immunoprecipitation. Lysates of spleen cells that had been labeled with [35S]methionine and [3H]histidine were precleared with rabbit anti-peptide serum reactive with the C-terminus of the canonical H-2Kb. The shortened form of H-2Kb was immunoprecipitated from this lysate with H-2Kb alloantiserum. Both forms of H-2Kb were isolated by NaDodSO4/PAGE. Tryptic peptide mapping confirmed that these molecules differed only at their C-terminus. The shortened form of H-2Kb is also found in a B-cell line (R8) but not in three cloned T-cell lines or in a T-cell lymphoma (EL4), suggesting that regulatory events are involved in producing the two forms of H-2Kb. Putative lariat branch points involved in these alternative splicing events for the 3' coding region of H-2 class I pre-mRNAs are proposed.