The relationship between rat major acute phase protein and the kininogens

The rat major acute phase protein (alpha 1-MAP) is a cysteine protease inhibitor. The stoichiometry of the interaction between the inhibitor and enzyme was shown to be 1:2. A cDNA clone specific for rat alpha 1-MAP was isolated from a cDNA library prepared from an inflamed rat liver RNA template. The 1458-base pair insert was sequenced and positively identified by alignment with a partial amino acid sequence obtained by radiosequence analysis of the primary translation product for alpha 1-MAP. Complete sequence analysis determined the alpha 1-MAP cDNA coded for the entire protein with the exception of the first four amino acids of the signal peptide, all of which were identified by radiosequencing. The coding sequence spans 1282 nucleotides, followed by 115 base pairs of a 3' untranslated region. Two putative active sites, suggested by the enzyme-inhibitor ratio, have been identified by analysis of internal duplications of the alpha 1-MAP sequence and the alignment of these regions with the sequences of several low molecular weight cysteine protease inhibitors. A computer homology analysis of the protein sequence revealed a 59.3% overall identity between rat alpha 1-MAP and bovine low molecular weight (LMW) kininogen. The homology included the signal peptide regions. LMW kininogen is a precursor of bradykinin. alpha 1-MAP does contain a bradykinin sequence; the flanking amino acids are different, however. Evidence for the expression of the LMW and a high molecular weight kininogen from the same gene, and the high degree of homology between these proteins and the rat acute phase protein suggest that all three proteins belong to a precisely regulated gene family.     

A set of U1 snRNA-complementary sequences involved in governing alternative RNA splicing of the kininogen genes

The rat K and T kininogen genes show different modes of mRNA production. The K gene encodes two distinct mRNAs for high molecular weight (HMW) and low molecular weight (LMW) kininogens. These two mRNAs are generated by differential usage of the 3'-terminal exon (LMW exon) and the exon next to and upstream from the LMW exon (HMW exon) through alternative splicing and polyadenylation. In contrast, the T gene generates one mRNA by using selectively the LMW exon, although the T gene is extremely homologous to the K gene. In this study, we constructed a series of chimeric kininogen genes by not only exchanging equivalent restriction fragments of the two genes but also replacing nucleotides that differ between the two genes. We then examined the sequences and the mechanisms governing the different expression patterns of the two genes by transfecting the chimeric genes into heterologous COS cells. The results indicated that the different expression patterns of the K and T genes are governed by two separate internal sequences of the HMW and LMW exons. The internal HMW sequence contains a set of five repetitive sequences, and these repetitive sequences are highly complementary to the 5' portion of U1 snRNA. Furthermore, the nucleotide differences in the U1 snRNA-complementary sequences between the K and T genes have marked effects on the relative formation of the HMW and LMW mRNAs; this indicates that the repetitive sequences complementary to U1 snRNA play a crucial role in determining the relative expression of the two mRNAs. Based on these findings, we discuss a novel mechanism for alternative RNA processing, in which splicing efficiency is controlled by the interaction of U1 small nuclear ribonucleoproteins and the U1 snRNA-complementary repetitive sequences of the kininogen pre-mRNA.     

Localization of DNA sequences governing alternative mRNA production of rat kininogen genes

The two types of the rat kininogen genes show different modes of mRNA production. The K gene encodes two distinct mRNAs for high molecular weight (HMW) and low molecular weight (LMW) kininogens. These two mRNAs are generated by differential usage of the 3'-terminal exon (LMW exon) and the one next to this exon (HMW exon) through alternative polyadenylation and splicing. In contrast, the two T genes selectively generate the LMW form of the mRNA, although the T genes are extremely homologous to the K gene, including the sequence (psi HMW region) corresponding to the HMW exon of the K gene. In this study, we constructed a series of chimeric kininogen genes by exchanging equivalent restriction fragments of the K and T genes and examined the sequences and the mechanisms governing the different expression patterns of the kininogen genes by introducing the chimeric genes into heterologous COS cells. The results indicate that the formation of the two forms of the mRNA is controlled by two separate 3' sequences of the kininogen genes. One is located within the internal sequence of the HMW/psi HMW region, whereas the other is within the LMW exon and its preceding region. Our data also suggest that the different expression patterns of the kininogen genes are primarily governed by differing splicing efficiency.     

Primary structures of the mRNAs encoding the rat precursors for bradykinin and T-kinin. Structural relationship of kininogens with major acute phase protein and alpha 1-cysteine proteinase inhibitor

Three types of cloned cDNA sequences for rat low molecular weight prekininogens were isolated and determined by molecular cloning and sequence analysis. The deduced amino acid sequences indicated that one, termed K-prekininogen, represents the counterpart of the known low molecular weight prekininogen present in other mammals, while the other two, called T-prekininogens, contain a novel T-kinin sequence which was recently identified from rat plasma. Although T- and K-prekininogens are highly homologous with each other, both of the T-prekininogens contain methionine, instead of arginine or lysine, as an amino acid preceding T-kinin and exhibit two consecutive amino acid deletions in the preceding region of T-kinin as compared with K-prekininogen. The former finding accounts for the previous observation of strong resistance of T-kininogens to cleavage with trypsin or kallikreins, while the latter finding has been explained by the structural analysis of genomic clones in which T-kinin-coding exon is contracted at its intron junction. A partial nucleotide sequence reported recently for the rat major acute phase protein (alpha 1-MAP) mRNA was found to be extremely related to the corresponding portion of the rat T-prekininogen mRNA. Furthermore, consistent with the previous report of the structural identity of major acute phase protein and alpha 1-cysteine proteinase inhibitor, kininogen closely resembles not only the former but also the latter in the amino acid compositions. The interrelationship among the triad of these proteins has been discussed.     

Structure and expression of two kininogen genes in mice

Kininogens serve dual functions by forming a scaffold for the assembly of the protein complex initiating the surface-activated blood coagulation cascade and as precursors for the kinin hormones. While rats have three kininogen genes, for mice, cattle, and humans only one gene has been described. Here, we present sequence and expression data of a second mouse kininogen gene. The two genes, kininogen-I and kininogen-II, are located in close proximity on chromosome 16 in a head-to-head arrangement. In liver and kidney, both genes are expressed and for each gene three alternative splice variants are synthesized. Two of them are the expected high and low molecular weight isoforms known from all mammalian kininogens. However, for both genes also a third, hitherto unknown splice variant was detected which lacks part of the high molecular weight mRNA due to splicing from the low molecular weight donor site to alternative splice acceptor sites in exon 10. The physiological functions of the six kininogen isoforms predicted by these findings need to be determined.     

Phylogenetic utility and evidence for multiple copies of elongation factor-1alpha in the spider genus Habronattus (Araneae: Salticidae)

In the continuing quest for informative genes for use in molecular systematics, the protein-coding gene Elongation factor-1alpha (EF-1alpha) has rapidly become one of the most prevalent "single-copy" nuclear genes utilized, particularly in arthropods. This paper explores the molecular evolutionary dynamics and phylogenetic utility of EF-1alpha in the salticid spider genus Habronattus. As has been reported for other arthropod lineages, our studies indicate that multiple (two) copies of EF-1alpha exist in Habronattus. These copies differ in intron structure and thus in size, making it possible to easily separate PCR amplification products. We present data for an intronless EF-1alpha copy for three Habronattus species. The presence of nonsense mutations and generally elevated rates of amino acid change suggest that this copy is evolving under relaxed functional constraints in Habronattus. A larger taxon sample (50 species plus outgroups) is presented for an EF-1alpha copy that includes both intron and exon regions. Characteristics of both regions suggest that this is a functional, orthologous copy in the species sampled. Maximum-likelihood relative-rate comparisons show that exon third codon sites are evolving more than 100 times as fast as second codon sites in these sequences and that intron sites are evolving about twice as fast as exon third sites. In combination, the EF-1alpha data provide robust, species-level phylogenetic signal that is largely congruent with morphologically well supported areas of Habronattus phylogeny. The recovery of some novel clades, and the unexpected fragmentation of others, suggests areas requiring further phylogenetic attention.     

Kaposi's sarcoma-associated herpesvirus K8 exon 3 contains three 5'-splice sites and harbors a K8.1 transcription start site

Kaposi's sarcoma-associated herpesvirus (KSHV) K8 and K8.1 open reading frames are juxtaposed and span from nucleotide (nt) 74850 to 76695 of the virus genome. A K8 pre-mRNA overlaps the entire K8.1 coding region, and alternative splicing of KSHV K8 and K8.1 pre-mRNAs each produces three isoforms (alpha, beta, and gamma) of the mRNAs. We have mapped the 5' end of the K8.1 RNA in butyrate-induced KSHV-positive JSC-1 cells to nt 75901 in the KSHV genome and have shown that exon 3 of the K8 pre-mRNA in JSC-1 cells covers most part of the intron 3 defined previously and has three 5'-splice sites (ss), respectively, at nt 75838, 76155, and 76338. Selection of the nt 75838 5'-ss dictates the K8 mRNA production and overwhelms the RNA processing. Alternative selection of other two 5'-ss is feasible and leads to production of two additional bicistronic mRNAs, K8/K8.1alpha and -beta. However, the novel bicistronic K8/K8.1 mRNAs translated a little K8 and no detectable K8.1 proteins in 293 cells. Data suggest that production of the K8/K8.1 mRNAs may be an essential way to control K8 mRNAs, especially K8alpha, to a threshold at RNA processing level.     

Conserved exon-intron organization in two different caerulein precursor genes of Xenopus laevis. Additional detection of an exon potentially coding for a new peptide

From a genomic library of Xenopus laevis, two genes coding for different preprocaeruleins have been isolated and sequenced. These correspond to the type I and type III precursors analyzed previously at the cDNA level [Richter, K., Egger, R. and Kreil, G. (1986) J. Biol. Chem. 261, 3676-3680]. The type III gene comprises eight exons; the type I apparently contains eight exons as well, of which six have been sequenced. The genetic information for the dekapeptide caerulein is present on small exons of 45 base pairs. The two genes are highly homologous in their 5'-flanking region, the exon/intron boundaries, and long stretches of intron sequences. A possible scheme for the evolution of this small family of genes through exon and gene duplications is presented. In the type I gene, in place of one of the caerulein exons, a potential exon with conserved splice sites was discovered. If expressed in some frog cells, this exon would code for a new peptide 60% homologous to caerulein.     

Simian immunodeficiency virus displays complex patterns of RNA splicing.

The human and simian immunodeficiency viruses encode at least six gene products that apparently serve regulatory functions. To evaluate the regulation of simian immunodeficiency virus gene expression at the level of RNA splicing, we used the polymerase chain reaction to amplify and clone cDNAs corresponding to a large array of mRNAs from infected cells. We identified mRNAs that used splice acceptor sites upstream of the initiator codons for tat, rev, vpr, nef, vif, and vpx, suggesting that these proteins may be expressed from different mRNAs. We also provide hybridization data suggesting that the same splice acceptor site may be used for both rev and env mRNAs. Furthermore, we isolated both tat and rev cDNAs that utilized three alternative splice acceptor sites at the start of coding exon 2, indicating that different versions of these proteins may be encoded. Finally, approximately 10 to 20% of simian immunodeficiency virus mRNAs spliced an intron from their untranslated 5' ends, and sequences contained within this intron constituted a portion of the tat-responsive TAR element. Thus, alternative pre-mRNA splicing adds a level of complexity to simian immunodeficiency virus expression, which may affect several levels of gene regulation.