Antifreeze protein pseudogenes.

Three members, 11-3, F2 and 5a, of the type-I antifreeze protein (AFP) multigene family in winter flounder were sequenced. All three belong to the subset of AFP genes that are linked, but irregularly spaced, and show significant differences from the functional genes in tandem repeats. 11-3 and F2 appear to be pseudogenes. Their intron, 3'-exon and 3'-flanking DNAs are similar to those of other AFP genes, but their 5'-exon is either missing or extensively modified, and has stop codons present in all three reading frames. Based on a comparison of intron sequences of family members, 11-3/F2 may represent a residual progenitor AFP gene which was duplicated after reaching pseudogene status. The third gene, 5a, is remarkable in having a 3'-exon that encodes an exceptionally long, Ala-rich sequence that lacks any semblance of the 11-amino acid repeats found in 11-3, F2 and functional AFP genes. 5a might also be a pseudogene, because its presumed TATA box appears to have mutated.     

Characteristics of a multicopy gene family predominantly consisting of processed pseudogenes.

The monoclonal antibody MOC-32 detected a 40 kDa protein in Western blot analysis. Immunological screening of an expression library of human SCLC cells with MOC-32 led to the isolation of overlapping cDNA clones. One of these, cHD4, was 1.0 kbp long and of about the same size as its corresponding mRNA. Preceded by an in phase stop codon, an open reading frame of 885 bp was present in cHD4 and a translational product of only 33 kDa could be calculated. Biochemical and immunological analysis established the relationship between the 40 kDa antigen and the isolated coding sequences and resolved the apparent discrepancy between the calculated molecular weight and the observed electrophoretic mobility. Nucleotide sequence comparison of cHD4 to the EMBL database revealed that cHD4 was nearly identical to a sequence claimed to encode a laminin binding protein. Southern blot and nucleotide sequence analysis indicated the presence of multiple copies of the gene in the human genome. At least five of these appeared to represent processed pseudogenes.     

A cDNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in Helicoverpa armigera: molecular characterization and expression analysis associated with pupal diapause

The diazepam binding inhibitor (DBI) or the acyl-CoA-binding protein (ACBP) is a 9-10 kDa highly conserved multifunctional protein that plays important roles in GABA(A) receptor activity regulation, lipid absorption and steroidogenesis in various organisms. To study the functions of DBI/ACBP in insect development or diapause, we cloned the cDNA from Helicoverpa armigera (Har) utilizing rapid amplification of cDNA ends (RACE). By homology search, Har-DBI/ACBP is conserved with the DBI/ACBPs known from other insects. Northern blot analysis showed that DBI/ACBP gene expressed in nonneural and neural tissues. RT-PCR combined Southern blot analysis revealed that DBI/ACBP mRNA in the brain of nondiapause individual was much higher than that in the brain of diapausing insects. At early and middle stages of 6th instar larvae, the level of DBI/ACBP mRNA was higher in the midgut of diapause type than that in nondiapause type and low at late 6th instar larval stage and early pupal stage in both types. In the prothoracic gland (PG), DBI/ACBP expression appeared at a high level at middle and late stages of 6th larval instar in both nondiapause and diapause types, and declined after pupation. In vitro experiments revealed that DBI/ACBP mRNA in PG could be stimulated by synthetic H. armigera diapause hormone (Har-DH), suggesting that Har-DH may stimulate the PG to produce ecdysteroids by the DBI/ACBP signal pathway. By in vitro assay, we also found that FGIN-1-27, which has similar functions to DBI/ACBP in ecdysteroidogenesis, could induce PG ecdysteroidogenesis effectively, suggesting that DBI/ACBP regulates biosynthesis of ecdysteroids in PG. Thus, DBI/ACBP indeed plays a key role in metabolism and development in H. armigera.     

Characterization of the functional gene and several processed pseudogenes in the human triosephosphate isomerase gene family.

The functional gene and three intronless pseudogenes for human triosephosphate isomerase were isolated from a recombinant DNA library and characterized in detail. The functional gene spans 3.5 kilobase pairs and is split into seven exons. Its promoter contains putative TATA and CCAAT boxes and is extremely rich in G and C residues (76%). The pseudogenes share a high degree of homology with the functional gene but contain mutations that preclude the synthesis of an active triosephosphate isomerase enzyme. Sequence divergence calculations indicate that these pseudogenes arose approximately 18 million years ago. We present evidence that there is a single functional gene in the human triosephosphate isomerase gene family.     

The snRNP E protein multigene family contains five pseudogenes with common mutations.

Sequence data from three previously-uncharacterized members of the snRNP E protein multigene family suggest that each is a non-transcribed processed pseudogene, even though one clone has the potential to code for a full-length protein with greater than 90% similarity to previously-characterized E protein cDNAs. Each of the newly-analyzed family members is without introns, contains a tract of polyadenylic acid residues, and is flanked by short direct repeats. In addition, the three sequences all contain point mutations that distinguish them from the E protein coding sequence. Seven point mutations are common to the three sequences described here and to two previously-described E protein pseudogenes. Although all of these mutations are transitions, only 5 of 7 could have been generated by deamination of methylated cytosines in inactive genes. Thus, the common mutations in the pseudogenes suggest an origin other than the expressed gene that we have described. Allelic variants for two of the pseudogenes were detected and repetitive elements are located near four of the five E protein pseudogenes that have been characterized.     

A new member of the plasma protease inhibitor gene family.

A 2.1-kb cDNA clone representing a new member of the protease inhibitor family was isolated from a human liver cDNA library. The inhibitor, named human Leuserpin 2 (hLS2), comprises 480 amino acids and contains a leucine residue at its putative reactive center. HLS2 is about 25-28% homologous to three human members of the plasma protease inhibitor family: antithrombin III, alpha 1-antitrypsin and alpha 1-antichymotrypsin. A comparison with published partial amino acid sequences shows that hLS2 is closely related to the thrombin inhibitor heparin cofactor II.     

Acyl-CoA-binding protein from cow. Binding characteristics and cellular and tissue distribution.

Hydroxyl radicals (OH.) in free solution react with scavengers at rates predictable from their known second-order rate constants. However, when OH. radicals are produced in biological systems by metal-ion-dependent Fenton-type reactions scavengers do not always appear to conform to these established rate constants. The detector molecules deoxyribose and benzoate were used to study damage by OH. involving a hydrogen-abstraction reaction and an aromatic hydroxylation. In the presence of EDTA the rate constant for the reaction of scavengers with OH. was generally higher than in the absence of EDTA. This radiomimetic effect of EDTA can be explained by the removal of iron from the detector molecule, where it brings about a site-specific reaction, by EDTA allowing more OH. radicals to escape into free solution to react with added scavengers. The deoxyribose assay, although chemically complex, in the presence of EDTA appears to give a simple and cheap method of obtaining rate constants for OH. reactions that compare well with those obtained by using pulse radiolysis.     

A genome-wide survey of human thioredoxin and glutaredoxin family pseudogenes

The thioredoxin/glutaredoxin family consists of small heat-stable proteins that have a highly conserved CXXC active site and that participate in the regulation of many redox reactions. We have searched the human genome sequence to find putative pseudogenes (non-functional copies of protein-coding genes) for all known members of this family. This survey has resulted in the identification of seven processed pseudogenes for human Trx1 and two more for human Grx1. No evidence for the presence of processed pseudogenes has been found for the remaining members of this family. In addition, we have been unable to detect any non-processed pseudogenes derived from any member of the family in the human genome. The seven thioredoxin pseudogenes can be divided into two groups: Trx1-psi2, -psi3, -psi4, -psi5 and -psi6 arose from the functional ancestor, whereas Trx1-psi1 and -psi7 originated from Trx1-psi2 and -psi6, respectively. In all cases, the pseudogenes originated after the human/rodent radiation as shown by phylogenetic analysis. This is also the case for Grx1-psi1 and Grx1-psi2, which are placed between rodent and human sequences in the phylogenetic tree. Our study provides a molecular record of the recent evolution of these two genes in the hominid lineage.     

Inhibition of rat tissue kallikrein gene family members by rat kallikrein-binding protein and alpha 1-proteinase inhibitor.

The regulation of tissue kallikrein activity by plasma serine proteinase inhibitors (serpins) was investigated by measuring the association rate constants of six tissue-kallikrein family members isolated from the rat submandibular gland, with rat kallikrein-binding protein (rKBP) and alpha 1-proteinase inhibitor (alpha 1-PI). Both these serpins inhibited kallikreins rK2, rK7, rK8, rK9 and rK10 with association rate constants in the 10(3)-10(4) M-1.s-1 range, whereas only 'true' tissue kallikrein rK1 was not susceptible to alpha 1-PI. This results in slow inhibition of rK1 by plasma serpins, which could explain why this kallikrein is the only member of the gene family identified so far that induces a transient decrease in blood pressure when injected in minute amounts into the circulation.