Sequence analysis of a cDNA clone encoding the C-terminal end of human complement factor H

Peptide sequencing of the complement system regulatory protein, factor H, permitted the synthesis of a mixed sequence oligonucleotide probe. Human liver cDNA libraries were screened and factor H-specific clones selected. No full-length clone was obtained, but the largest available clone, R2a, was found to encode the C-terminal 657 amino acids of factor H. The derived amino acid sequence consists of 10 contiguous internally homologous segments, each about 60 amino acids long. Sequences homologous to these are found in several other complement and non-complement proteins. Such sequences are likely to represent a particular type of tertiary structure subunit.     

Cloning of the am (glutamate dehydrogenase) gene of Neurospora crassa through the use of a synthetic DNA probe

In a previous study the alteration in the amino acid sequence of Neurospora crassa NADP-specific glutamate dehydrogenase (GDH) resulting from two mutually compensating frameshift mutations was used to deduce the first 17 nucleotides of the coding sequence of the am gene. In the work reported here, a synthetic 17-mer corresponding to the deduced sequence was shown to hybridize strongly to a 9-kb HindIII fragment from N. crassa wild-type DNA but not to any corresponding fragment from the DNA of a mutant strain known to be deleted for most or all of the gene. Wild-type HindIII fragments were fractionated for size and a fraction centering around 9 kb was cloned in vector λL47. Two clones carrying the strongly hybridizing fragment were identified. The hybridization to the 17-mer was localized within a 2.7-kb BamHI fragment and, within this, to a 700-bp BamHI-BglII subfragment. 5' end-labelled polyadenylated RNA isolated from wild-type mycelium hybridized to the 2.7-kb BamHI fragment and not appreciably to flanking fragments. The partial sequence analysis of the BamHI-BglII fragment has confirmed that the 17-mer probe matches the coding sequence at the 5' end of the gene and has also revealed an intervening sequence 67 bp in length, interrupting codon 15. Both the 9-kb HindIII fragment and the 2.7-kb BamHI fragment have been shown to be capable of transforming the deletion mutant to prototrophy and ability to produce GDH. Analysis of one transformant showed that the am gene was integrated, together with a part of the long arm of the lambda vector, at an unusual locus. This transformant, in which the am gene does not show its normal linkage to the linkage group 5 marker inl, was found to produce GDH to about 20% of the normal level.     

Cloning and analysis of the gene encoding lectin from the acorn barnacle Megabalanus rosa

In the acorn barnacle Megabalanus rosa, two types of galactose-binding C-type lectins (BRA-2 and BRA-3) have been identified. Here, we report the isolation and characterization of BRA-2 cDNA and genomic clones. In contrast to the BRA-3 gene, which consists of four exons, BRA-2 is encoded by a single exon, implying differences in the physiological roles of the two lectins.     

Two mRNAs exist for the Hs PROS-30 gene encoding a component of human prosomes.

Screening of a λgt11 cDNA expression library of the HeLa cell genome with a monoclonal antibody that specifically recognizes prosomal 30-33-kDa proteins, allowed isolation of a 1264-nucleotide (nt) recombinant cDNA containing a 327-nt untranslated 5'-end. The amino acid (aa) sequence deduced from this cDNA revealed a protein of 269 aa (M_r of 30 227) that includes a consensus box characteristic for Tyr phosphorylation, also observed in other prosomal proteins. Comparison with another prosomal 27-kDa protein, cloned in our laboratory, indicated the presence of three prosomespecific homology boxes observed in these proteins from archaebacteria to man. Interestingly, except for the untranslated 5'-end, as well as the sequence coding for the N-terminal six aa, this cDNA is identical to two recently published cDNAs encoding subunit C2 of human liver proteasome [Tamura et al., Biochim. Biophys. Acta 1089 (1991) 95–102] and subunit NU of human erythrocyte macropain [DeMartino et al., Biochim. Biophys. Acta 1079 (1991) 29–38]. Primer extension and Northern blot analysis using two specific 18-mer oligodeoxyribonucleotides indicated the presence of two mRNAs that have divergent 5'-ends. These results, as confirmed by the polymerase chain reaction, establish the existence of two distinct Hs PROS-30 mRNAs, differing in their 5'-noncoding regions and in the N-terminal six aa of their protein products.     

The Drosophila PROS-28.1 gene is a member of the proteasome gene family

In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-M_r complex possessing proteolytic activity. Screening a Drosophila λgt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene.     

Structure and expression of a gene from Arabidopsis thaliana encoding a protein related to SNF1 protein kinase

The AKin10 gene from Arabidopsis thaliana encoding a putative Ser/Thr protein kinase (PK) has been isolated and characterized. The AKin10-encoding gene is located on a genomic 5.4-kb BamHI fragment and contains ten introns, one being located in the 5' untranslated region. The deduced amino acid sequence of AKin10 is 65% identical over the catalytic domain to the yeast PK (SNF1). SNF1 is essential for the derepression of many glucose-repressible genes, including Suc2 which encodes invertase. Southern blot hybridization experiments suggested the presence of one copy of the gene per haploid genome of A. thaliana. Northern hybridization experiments indicated that this gene is expressed in roots, shoots and leaves. AKin10 may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism in higher plants.     

Nucleotide sequence of the VP1 gene of the foot-and-mouth disease virus strain A Venceslau

The VP1 coat protein of FMDV strain A Venceslau (Aven) consists of 213 amino acid residues. Serum neutralization tests demonstrated that strain Aven is closely related to strain A Argentina/79 (A₇₉) but significantly different from strain A₂₄ Cruzeiro (A₂₄). There is a strong correlation between the amino acid sequences and the serological data. Nucleotide and amino acid sequence analyses of VP1 showed that serologically related viruses (Aven and A₇₉) differ less in this region of the genome than those of serologically distinct viruses (Aven vs. A₂₄). The most significant variation between Aven and A₂₄ occurs at amino acid positions 43 to 46, in which all four residues are different.     

Molecular analysis of the human interferon-alpha gene family

Fifteen DNA clones containing sequences related to human interferon-alpha cDNA were isolated from a human chromosomal gene bank (Nagata et al., Nature 287 (1980) 401-408) and characterized by restriction mapping, R-loop and heteroduplex analysis. Nine distinct DNA segments hybridized strongly with interferon-alpha 1 cDNA and formed R-loops with poly(A) RNA from interferon-producing human leukocytes; most if not all of these segments represent functional interferon genes. Five segments hybridized weakly with the probe and did not form R-loops with the poly(A) RNA; one of these was characterized as an interferon-alpha pseudogene. Several DNA segments overlap and define a region of 36 kilobase pairs (kb) that contains three strongly and three weakly hybridizing sequences. From our data and those of Goeddel et al. (Nature 290 (1981) 20-25) we conclude that there exist at least 11 distinct genes of gene-like sequences of the interferon-alpha type in the human genome, of which most likely represents an allelic variant, and at least five pseudogenes distantly related to the interferon-alpha genes.     

Molecular analysis of the gene encoding a protein component of the Eikenella corrodens adhesin complex that is close to the carbohydrate recognition domain

A monoclonal antibody against a lectin-like substance (LS) of Eikenella corrodens (Ec) was used for screening the Ec DNA library. Three positive clones that carried an identical 12-kb segment were obtained. A 25-kDa protein, which specifically binds to the antibody, was overproduced in all of the Escherichia coli clones. Deletion analysis showed that the gene encoding the 25-kDa protein was located within a 1.2-kb segment. The nucleotide (nt) sequence of this segment contained an open reading frame encoding a protein of 24 600 Da. We purified the 25-kDa protein from the cloned E. coli strain. The sequence of the first 10 amino acids (aa) from the N-terminus of the purified 25-kDa protein agreed with that deduced from the nt sequence. Since the monoclonal antibody used in this study inhibits the physiological activity of EcLS, we concluded that the 25-kDa protein is a component of the adhesin complex, which is located near the carbohydrate recognition domain of lectin in EcLS.     

Cloning and characterization of the gene cluster encoding type 3 (MR/K) fimbriae of Klebsiella pneumoniae

Abstract The gene cluster encoding the type 3 fimbriae of a Klebsiella pneumoniae isolate was cloned using the cosmid-cloning technique. Escherichia coli transformants, expressing type 3 fimbriae, were selected by reactivity with a monoclonal antibody directed against an epitope of the purified type 3 fimbriae. The phenotypic expression of type 3 fimbriae by transformants possessing the parental plasmid was dependent upon the host strain used. However, subcloning of this plasmid resulted in the construction of a chimeric molecule which imparted a stable phenotype regardless of the host strain. In addition, subcloning of the parental recombinant plasmid suggested that the minimal size of DNA necessary for production and expression of fimbriae was approximately 5.5 kb.     

Cloning and characterization of KM190, a specific satellite DNA family of Drosophila kitumensis and D. microlabis

The nucleotide sequences of nine clones, pKA191/l-4 from Drosophila kitumensis and pMR.190/1–5 from D. microlabis, were determined. They represent a tandemly arranged and highly repetitive satellite DNA family, KM190, which is specific for the two species.     

Nucleotide sequence of the transposable element IS15

We have determined the complete nucleotide sequence of the right (R) copy of the insertion sequence IS15 which flanks, in direct orientation, the composite transposon Tn1525. IS15-R, which is capable of independent transposition, is 1648 bp long and has short (14 bp) perfect inverted repeats at its termini. Analysis of the nucleotide sequence indicates that IS15-R results from the transposition, in direct orientation, of a smaller (820 bp long) IS, designated IS15-Δ, into itself. This integration event is accompanied by the duplication of 8 bp in the target DNA. IS15-Δ possesses two large overlapping open reading frames (ORF) located on opposite strands. Because of this particular structure, IS15 possesses four large ORFs which, due to the integration event, exhibit some differences with those of the parental 1S15-Δ.     

Sequence complexity of the S receptor kinase gene family in Brassica

A genomic library from an S ₂₉/S ₂₉ self-incompatible genotype of Brassica oleracea was screened with a probe carrying part of the catalytic domain of a Brassica S-receptor kinase (SRK)-like gene. Six positive phage clones with varying hybridisation intensities (K1 to K6) were purified and characterised. A 650–700 by region corresponding to the probe was excised from each clone and sequenced. DNA and predicted protein sequence comparisons based on a multiple alignment identified K5 as a pseudogene, whereas the others could encode functional proteins. K3 was found to have lost an intron from its genomic sequence. The six genes display different degrees of sequence similarity and form two distinct clusters in a dendrogram. The 98% similarity between K4 and K6, which extends across intron sequences, suggests that these might be very recently diverged alleles or daughters of a duplication. In addition, K2 showed a comparably high similarity to the probe. Clones K1, K3 and K5 cross-hybridised with an SLG ₂₉ cDNA probe, indicating the presence of upstream receptor domains homologous to the Brassica SLG gene. This suggests that the previously reported S sequence complexity may be ascribed to a large receptor kinase gene family.