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The molecular basis for electrophoretic variations of rat alpha-fetoprotein was studied. Stepwise deglycosylations of proteins and radiolabeling of the sugars indicated that the number of such chains per molecule is two and one for the slow and fast variants, respectively.
The complete 4775-nt cDNA encoding the human serotonin 5-HT2c receptor (5-HT2cR), a G-protein-coupled receptor, has been isolated. It contains a 1377-nt coding region flanked by a 728-nt 5'-untranslated region and a 2670-nt 3'-untranslated region. By using the cloned 5-HT2cR cDNA probe, the complete human gene for this receptor has been isolated and shown to contain six exons and five introns spanning at least 230 kb of DNA. The coding region of the human 5-HT2CR gene is interrupted by three introns, and the positions of the intron/exon junctions are conserved between the human and the rodent genes. In addition, an alternatively spliced 5-HT2CR RNA that contains a 95-nt deletion in the region coding for the second intracellular loop and the fourth transmembrane domain of the receptor has been identified. This deletion leads to a frameshift and premature termination so that the short isoform RNA encodes a putative protein of 248 amino acids. The ratio for the short isoform over the 5-HT2CR RNA was found to be higher in choroid plexus tumor than in normal brain tissue, suggesting the possibility of differential regulation of the 5-HT2CR gene in different neural tissues or during tumorigenesis. Transcription of the human 5-HT2CR gene was found to be initiated at multiple sites. No classical TATA-box sequence was found at the appropriate location, and the 5'-flanking sequence contains many potential transcription factor-binding sites. A 7.3-kb 5'-flanking 5-HT2CR DNA directed the efficient expression of a luciferase reporter gene in SK-N-SH and IMR32 neuro-blastoma cells, indicating that it contains a functional promoter.
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A variant clone of Snyder-Theilen feline sarcoma virus (ST-FeSV) encoding a polyprotein with a molecular weight of approximately 104 kDa (P104) was compared to the P85 encoding prototype clone of ST-FeSV. Analysis of chimeric genes constructed with the viral oncogenes of the two clones indicated that the variant clone coded for a larger polyprotein than the prototype clone because of genetic differences in its 3' portion. Comparative DNA sequence analysis revealed that one nucleotide just upstream of the termination condon TGA in the prototype proviral DNA was deleted from the variant clone resulting in a 468-bp larger open reading frame. Furthermore, it appeared that the U3 regions of the long terminal repeats (LTRs) of the variant clone contained an insertion of 71 bp as compared to the LTRs of the prototype clone. In addition, both clones differed also from each other with respect to genetic sequences deleted from their env gene regions.
Soybean (Glycine max L.) storage proteins are composed mainly of two major components, beta-conglycinin and glycinin. Electrophoretic variants of the beta subunit of beta-conglycinin and the A3 polypeptide of glycinin were detected on SDS-PAGE, and designated them as beta* and A3*, respectively. beta* and A3* exhibited higher and lower mobilities, respectively, than the common beta subunit and A3 polypeptide. The N-terminal nine and 10 amino acid sequences of beta* and A3* were completely identical to the previously reported sequences of the beta subunit and the A3 polypeptide, respectively. Analysis using concanavalin A-horseradish peroxidase and treatment with N-glycosidase indicated that glycans were not responsible for the difference in electrophoretic mobility of beta* or A3*. Furthermore, five clones of beta* or beta and three clones of A3*, respectively, were sequenced but we could not detect deletions and insertions except for a single or a few amino acid substitutions as compared with the common beta subunit and A3 polypeptide. These results indicate that a single or a few amino acid substitution affects the electrophoretic mobilities of beta* and A3*.
NMR has been used to examine the conformational properties of two variants of the sarcin-ricin loop (SRL) from eukaryotic 28S rRNA, which is essential for elongation factor interactions with the ribosome: (1) its bacterial homologue, which lacks two of the bases that flank the conserved 12-nt sequence in the middle of the SRL, but which is functionally equivalent, and (2) a functionally active variant of the eukaryotic SRL in which the bulged G within the conserved sequence is replaced by an A. The data indicate that, although the bacterial SRL is less stable than the eukaryotic SRL, its conformation is closely similar. Furthermore, even though replacement of the bulged G in the SRL with an A seriously destabilizes the center of the loop, its effect on the overall conformation of the SRL appears to be modest. In the course of this work, it was serendipitously discovered that at neutral pH, the C8 proton of the bulged G, in both PRO-SRL and E73, exchanges about 10 times faster than it does in GMP.
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Wild populations of Drosophila mojavensis exhibit considerable conformational variation in the NAD+-free form of alcohol dehydrogenase (alcohol:NAD+ oxidoreductase; EC 1.1.1.1). The variation appears genetic, as it does not occur within an inbred strain. The NAD+-bound form of alcohol dehydrogenase, present in the same individuals, does not exhibit the variation, suggesting that the binding of NAD+ acts to stabilize conformation. Such cofactor binding to enzymes may thus conceal considerable variation. A similar effect is suggested for binding of esterase subunits.
Superoxide dismutase (SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) is known to be polymorphic in many organisms; in Drosophila, the degree of polymorphism has a wide range of variation from locality to locality within a given species. We have thoroughly purified from D. melanogaster the two common electromorphs, SODS and SODF. These differ in properties such as isoelectric point, specific activity, rate constant, thermostability, and amino acid composition. The specific activity is three times greater in SODS than in SODF, but the latter is more thermostable. In strains from California, SODS differs from SODF by at least one amino acid substitution: lysine in SODS is replaced by either aspartic acid or asparagine in SODF. This difference is consistent with the electrophoretic mobility and isoelectric points of the two electromorphs. In strains from Africa, SODS and SODF differ by two amino acid substitutions (histidine and proline in SODS vs. serine and either glutamic acid or glutamine in SODF) in addition to the one distinguishing the California strains. Thus the SODF electromorphs from California and from Tunisia, in spite of their identical electrophoretic mobility, differ by at least two amino acid substitutions.
We report the emergence of drug-resistant viral mutations in chronically HIV-infected individual undergoing structured treatment interruptions (STI). THe protease mutations K101E and K103N were detected at the end of the second or third STI. We concluded that the repeated abrupt termination and resumption of certain antiretroviral drug regimens during STI therapy may lead to the development of drug resistance in chronically HIV-infected individuals.
The amino acid sequences of the two variants (H1a 121 residues and H1b 119 residues) of the sperm-specific histone H1 from the polychaete annelid Platynereis dumerilii have been completely established. Comparison of the sequences of these two variants shows one deletion of two residues in histone H1b and 22 substitents, of which most occur in the globular domain. The two variants differ highly in a sequence of nine residues adjacent to the conservative phenylalanine residue of histone H1 (64-72 in H1a, 62-70 in H1b) which makes H1a less hydrophobic than H1b. The small molecular size of Platynereis H1a and H1b is a unique feature among the histones H1 of which the size ranges between 189 residues (chicken erythrocyte H5) and 248 residues (sea urchin sperm H1). H1a and H1b have short N- and C-terminal basic domains but the size of the globular domain (approximately equal to 80 residues) is similar to that of other H1s. In the globular region the variant H1a exhibits a close relationship with somatic or sperm H1s whereas the variant H1b is more related to H5 histones.
High-performance liquid chromatography allows one to perform effective and rapid separations on microquantities of material. The miniaturization of the techniques led to new approaches involving isoelectric focusing (IEF) of immobilized pH gradient IEF for haemoglobin preparation, successive enzymic hydrolyses for isolating abnormal peptides and amino acid composition determination after precolumn derivatization.
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BRCA1 and BRCA2 are tumor suppressor genes shown to be involved in 90% of familial breast cancers and also known to be involved in ovarian and prostate cancers. Both BRCA1 and BRCA2 gene products are regulated in a cell cycle-dependent manner and have potential transactivation function. Here, we show that BRCA2 undergoes differential splicing giving rise to a novel variant protein BRCA2a, lacking putative transcriptional activation domain. Both BRCA2a and BRCA2 are expressed at high levels in thymus and testis but moderate levels in mammary gland and prostate suggesting that BRCA2a and BRCA2 may have a role in the development and differentiation of these tissues.
The catalytic subunit of human DNA polymerase epsilon, an enzyme involved in nuclear DNA replication and repair, is encoded by the POLE1 gene. This gene is composed of 51 exons spanning at least 97 kb of genomic DNA. It was found to encode three alternative mRNA splice variants that differ in their 5'-terminal sequences and in the N-termini of the predicted proteins. A CpG island covers the promoter region for the major transcript in HeLa cells. This promoter is TATA-less and contains several putative binding sites for transcription factors typical of S-phase-up-regulated and serum-responsive promoters. Potential promoter regions were also identified for the two other alternative transcripts. Interestingly, no nuclear polyadenylation signal sequence was detected in the 3'-untranslated region, although a poly(A) tail was present. These results suggest a complicated regulatory machinery for the expression of the human POLE1 gene, including three alternative transcripts expressed from three promoters.
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The zonal study of the immunological structure of the population revealed an increase in the number of infective agents with which the population comes into contact in the southern regions. In the medial boreal forest zone contacts of low intensity only with tick-borne encephalitis virus were detected (the immune stratum was found to be 5.5%, 2,178 persons were examined); in the southern boreal forest zone the foci of tick-borne encephalitis actively manifested (the immune stratum reached 38.9%, 2,854 persons were examined), and Q fever was constantly detected (1.6%). In the forest-steppe zone the population was found to have contacts with the causative agent of tick-borne encephalitis (the immune stratum was found to be 13.3%, 2,056 persons were examined), Q fever (1.2%) and tick-borne rickettsiosis (1.1%).