A reevaluation of the amino acid sequence of human follitropin beta-subunit

A collaborative study from two laboratories has been undertaken to re-evaluate the human follitropin -subunit sequence (hFSH), since areas of uncertainty remain in the wake of two earlier reports. The first report was by Shome and Parlow (1974). The second, by Saxena and Rathnam (1976), proposed revisions for sequence not definitively placed in the first study, as well as some differences in other placements. We have re-examined the sequence of the hFSH with more recent methodology. This has led to revision of certain areas of the sequence and resolution of differences between the two earlier proposals. Specifically, an-Ile-Ser- is established at 21–22, Asp at 41, Arg at 44, Lys at 46, and Glu at 111. These were areas of disagreement in the earlier proposals. A definitive placement of the residues around tryptophan-27 has now been obtained by three laboratories. C-terminal heterogeneity was observed with subunits ending at residue 107, 109, or 111. N-terminal heterogeneity has been observed in all preparations examined to date. A significant population of molecules with a proteolytic nick between residues 38–39 is noted. This is very likely an artifact of the collection and processing. The preparations examined in the present studies showed no evidence of residues 112–118 proposed by Saxena and Rathnam.     

Analysis of the Escherichia coli glycogen gene cluster suggests that catabolic enzymes are encoded among the biosynthetic genes

The nucleotide sequences of the Escherichia coli genome between the glycogen biosynthetic genes glgB and glgC, and 1170 bp of DNA which follows glgA have been determined. The region between glgB and glgC contains an open reading frame (ORF) of 1521 bp which we call glgX. This ORF is capable of coding for an M_r 56 684 protein. The deduced amino acid (aa) sequence for the putative product shows significant similarity to the E. coli glycogen branching enzyme, and to several different glucan hydrolases and transferases. The regions of sequence similarity include residues which have been reported to be involved in substrate binding and catalysis by taka-amylase. This suggests that the proposed product may catalyze hydrolysis or glycosyltransferase reactions. The cloned region which follows glgA contains an incomplete ORF (1149 bp), glgY, which appears to encode 383 aa of the N terminus of glycogen phosphorylase, based upon sequence similarity with the enzyme from rabbit muscle (47% identical aa residues) and with maltodextrin phosphorylase from E. coli (37% identical aa residues). Results suggest that neither ORF is required for glycogen biosynthesis. The localization of glycogen biosynthetic and degradative genes together in a cluster may facilitate the regulation of these systems in vivo.     

Cloning and sequencing of the gene encoding flavodoxin from Desulfovibrio vulgaris Hildenborough

Abstract The gene encoding flavodoxin from Desulfovibrio vulgaris Hildenborough (148 amino acid residues), the first flavoprotein for which a three-dimensional structure has been determined, was cloned with the use of two synthetic oligonucleotides, designed to recognize the coding sequence for amino acid residues 11–19 and 98–103, respectively. The two oligonucleotides were used to screen a library of 900 λ-clones of the D. vulgaris chromosome. A single clone, λFL1, reacting with both probes was isolated. The entire structural gene for flavodoxin is contained in the 15 kb insert of λFL1 as found by nucleic acid sequencing. The codon usage in the flavodoxin gene is strongly biased towards G or C in the third codon position. A table in which codon usage information from all genes of D. vulgaris sequenced to date is combined is presented and should facilitate further gene cloning with oligonucleotide probes.     

Biochemical similarity among serologically distinct flagellins of Campylobacter jejuni

Abstract We describe a simplified method for obtaining highly purified flagellin, suitable for biochemical analysis using HPLC-gel permeation. Amino acid composition and N-terminal sequence analyses were performed on flagellins from serologically distinct isolates. The amino acid composition of flagellin from 10 strains was very similar. The N-terminal amino acid sequence is highly conserved. Significant sequence homology was found with flagellin of Bacillus subtilis .     

Frequent duplication and deletion events in the 5S RNA genes and the associated spacer regions of theTriticeae

The 5 S DNA units from 15 grasses in theTriticeae were analysed at the DNA sequence level. Four units carried duplications near the 3-end of the 5 S RNA gene with 3 of the duplications centred on the same base pairs as a duplication previously reported byGerlach & Dyer. The fourth duplication was located 3 downstream from the gene, in the spacer region. Apparent deletions were very frequent when units of the different grasses were compared and it was clear that these deletions did not extend into a 75 bp spacer region upstream from the 5 S RNA gene. This 75 bp region also tended to be more conserved between the grasses as compared to the high level of sequence change in the rest of the spacer region. — Phenetic relationships were established between the grasses using the sequence data. The relationships were generally consistent with the data from other parameters and, in addition, showed that two Australian grasses were closely related to the other Northern hemisphere genera examined. The data concerning the Australian grasses is discussed in relation to the isolated nature of Australia.     

The synthetic precursor specific region of pre-pro-parathyroid hormone forms ion channels in lipid bilayers

We have used the chemically synthesized sequence of pre-pro-parathyroid hormone and several of its analogues to test the notion that the capacity of amphipathic peptides to aggregate in membranes and form ion-permeable channels correlates with their ability to function as signal sequences for secreted proteins. We found that pre-pro-parathyroid hormone (the signal sequence and pro-region of parathyroid hormone (M)), as well as some of its analogues, forms aggregates of monomers which are ion-permeable. The ion-permeable aggregates (2–3 monomers) formed by (M) are voltage-dependent and are more permeable for cations than for anions. The compounds which formed ion channels in bilayers also acted as potential signal sequences. We conclude that the ability of peptides to form ion-permeable pathways in bilayers may be correlated to their ability to function as signal peptides.     

Molecular characterization of the virulence gene virA of the Agrobacterium tumefaciens octopine Ti plasmid

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. Induction of vir gene expression by plant signal molecules is solely dependent on the virulence loci virA and virG. This study focused on the virA locus of the octopine type Ti plasmid pTi15955. The nucleic acid sequence of a 5.7-kilobase fragment encompassing virA was determined. Genetic analysis of this region revealed that virA contains one open reading frame coding for a protein of 91 639 daltons. Immunodetection with antibodies raised against a 35-kDa VirA fusion protein produced in E. coli identified the VirA product in wild-type Agrobacterium cells. Moreover, it is shown that the VirA protein is located in the cytoplasmic membrane fraction of Agrobacterium. These data confirm the proposed regulatory function of VirA whereby VirA acts as a membrane sensor protein to identify plant signal molecules in the environment. The proposed sensory function of VirA strikingly resembles the function of the chemotaxis receptor proteins of E. coli.     

Four genes in two diverged subfamilies encode the ribulose-1,5-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana

The multigene family encoding the small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase/oxygenase in the crucifer Arabidopsis thaliana has been isolated and the organization and structure of the individual members determined. The family consists of four genes which have been divided into two subfamilies on the basis of linkage and DNA and amino acid sequence similarities. Three of the genes, designated ats1B, ats2B, and ats3B, reside in tandem on an 8 kb stretch of the chromosome. These genes share greater than 95% similarity in DNA sequence and encode polypeptides identical in length and 96.7% similar in amino acid sequence. The fourth gene, ats1A, is at least 10 kb removed from, or completely unlinked to the B subfamily. The B subfamily genes are more similar to each other than to ats1A in nucleotide and amino acid sequence. All four genes are interupted by two introns whose placement within the coding region of the genes is conserved. The introns of the B subfamily genes are similar in length and nucleotide sequence, but show no similarity to the introns of ats1A. Comparison of the DNA sequences within the immediate 5 and 3 flanking sequences among the genes revealed only limited regions of homology. S1 analysis shows that all four genes are expressed.