The nature of the lamprey visual pigment

From the retina of the land-locked population of the sea lamprey, Petromyzon marinus, a photolabile pigment was extracted which was identified spectrophotometrically as a member of the rhodopsin group of pigments. Using the absorption spectrum of a relatively pure solution and analysis by means of difference spectra, the peak of this pigment was placed at about 497 mµ. The method of selective bleaching by light of different wave lengths revealed no significant amounts of any other pigment in the extracts. A similar pigment was also detected in retinal extracts of the Pacific Coast lamprey, Entospenus tridentatus. These results are significant for two reasons: (a) the lamprey is shown to be an example of an animal which spawns in fresh water but which is characterized by the presence of rhodopsin, rather than porphyropsin, in the retina; (b) the primitive phylogenetic position of the lamprey suggests that rhodopsin was the visual pigment of the original vertebrates.     

Isolation and characterization of lamprey rhodopsin cDNA.

Genomic DNA fragments coding a visual pigment of the lamprey were amplified by polymerase chain reaction, using oligonucleotide mixtures as primers. The complete coding region of the cDNA was obtained by separate amplification of both cDNA ends. The deduced amino acid sequence of the coding region showed 78-82% identity with those of rhodopsins of higher vertebrates, but only 43-47% identity with those of human color pigments. The cloned DNA appears to be the cDNA of a lamprey rhodopsin, which is expressed in the "short" photoreceptor cell.     

Molecular cloning of the common carp (Cyprinus carpio) rhodopsin cDNA

A recombinant phage clone containing a 1584 nucleotides rhodopsin cDNA was screened from a carp retinal cDNA library. The inserted DNA consisting of a single open reading frame of 1062 nucleotides at positions 72 to 1133 encodes a 354 amino acid polypeptide. The deduced amino acid sequence of carp rhodopsin showed 95.7, 85.5 and 74.4% identity with that of goldfish, sand goby and lamprey, respectively. The sites of palmitoylation, glycosylation, disulfide bond formation and Schiff base formation in the putative rhodopsin are all conserved.     

Cloning,sequencing and analysis of the ggh-A gene encoding a 1,4-β-d-glucan glucohydrolase from Microbispora bispora

The ggh-A gene, encoding a 1,4-β-d-glucan glucohydrolase/β-glucosidase, of Microbispora bispora (Mb) was subcloned and expressed from a 4.0-kb XhoI DNA fragment. The nucleotide sequence of this fragment was determined. Analysis of the sequence revealed one open reading frame (ORF) which encodes a 986-amino-acid (aa) protein with a calculated molecular weight of 107 510. The ggh-A ORF has features typical of an actinomycete gene including high GC content (70.5%) and corresponding biased codon usage. Comparison of the aa sequence of the Mb 1,4-β-d-glucan glucohydrolase (Mbggh-A) with other glycosidases reveals high overall homology to several β-glucosidases and a 1,4-β-d-glucan glucohydrolase belonging to the glycosyl hydrolase family 3. The aa sequence alignments of Mbggh-A and β-glucosidases show that the active site region potentially involves two Asp residues. The aa sequence homology studies revealed a potential two-domain structure for Mbggh-A and other β-glucosidases. Furthermore, Mbggh-A has localized homology to a cellulose-binding domain present in some xylanases. This report is significant, as, to date, 1,4-β-d-glucan glucohydrolases have rarely been reported, though they are assumed to have a critical role in cellulolysis.     

Molecular cloning and sequencing of the gene encoding an exopolygalacturonase of a Bacillus isolate and properties of its recombinant enzyme

An exopolygalacturonase (exo-PGase; EC 3.2.1.82) was found in the culture broth of a Bacillus isolate. The gene encoding the exo-PGase, pehK, was cloned by polymerase chain reaction using mixed primers designed from N-terminal and internal amino acid (aa) sequences of the enzyme (PehK). The determined nucleotide (nt) sequence of pehK revealed a 2940 bp open reading frame (980 aa) that encoded a putative signal sequence (27 aa) and a mature protein (953 aa; 103 810 Da). The recombinant enzyme was purified to homogeneity from a culture broth of Bacillus subtilis harboring a pehK-containing plasmid. It had a molecular mass of 105 kDa and a pI value of 5.0. The maximum activity was observed at pH 8 and 55°C in Tris–HCl buffer. The degradation products from polygalacturonic or oligogalacturonic acids were digalacturonic acid, like the exo-PGases, PehX of Erwinia chrysanthemi and PehB of Ralstonia solanacearum. The deduced aa sequence of PehK exhibited moderate homology to those of PehX and PehB with approx. 30% identity for both. High homology was observed in a suitably aligned internal region of the three enzymes (65% identity), and some of the conserved aa residues appeared to form the catalytic core of the enzymes.     

Cloning and analysis of a cDNA encoding a two-domain hemoglobin chain from the water flea Daphnia magna

A cDNA encoding a two-domain hemoglobin (Hb) chain of Daphnia magna was cloned and its nucleotide (nt) sequence of 1261 bp was determined. The nt sequence contained 74 bp of the leader sequence, 1047 bp of an open reading frame (ORF), and 119 bp of the 3′-untranslated region (UTR), excluding the polyadenylation tail. A sequence, AATACA, located 24 bp upstream from the polyA sequence was considered to be a polyadenylation signal. cDNA-derived amino acid (aa) sequence revealed that D. magna Hb chain is synthesized as a secretory precursor with a signal peptide of 18 aa. Mature D. magna Hb chain consists of 330-aa residues with a calculated molecular weight of 36 227, which is composed of two large repeated domains, domain 1 and 2. Several key aa that are invariant in all or most of other Hb and required for functional heme-binding are conserved in each of the two domains. The N-terminal extension (pre-A segment) of domain 1 was unusually long and contained an unusual threonine-rich sequence. The homology between the aa sequences of the two domains (24% identity) was much lower than that observed in other two-domain Hb chains from clams or nematode. Hb mRNA level in D. magna reared under low oxygen concentration was more than 12 times higher than that in D. magna reared with sufficient aeration, indicating that the expression of Hb gene is regulated by mRNA level.     

Drosophila melanogaster P1 genomic clone DS05563 contains the chaperonin-encoding gene Cctg

We report the sequence analysis of a Drosophila melanogaster (Dm) P1 genomic clone (DS05563) which contains the γ-chaperonin-encoding gene, Cctg. The (Hs) Cctg orthologue was found to share strong sequence identity with a 1603-bp region of DS05563, suggesting that Dm Cctg is located within this region. Detailed analysis has shown that Dm Cctg comprises four exons and is interrupted by three introns of 55, 85 and 66 bp. Dm Cctg encodes a predicted peptide of 545 amino acids (aa) (approx. 60 kDa). The predicted Dm CCTγ aa sequence shares a high degree of sequence identity with γ-orthologues from human (70%), mouse (70%), protozoa (60%) and yeast (60%), and also contains domains found in other chaperonins including bacterial GroEL, mitochondrial Hsp60 and plant Rubisco large subunit-binding protein. These data support the conclusion that the DS05563 clone contains the Dm Cctg gene.     

Molecular cloning of a putative serotonin receptor gene from barnacle,Balanus amphitrite

We isolated a putative serotonin receptor gene from a genomic library of the barnacle, Balanus amphitrite Darwin, using an NcoI fragment of the barnacle G protein-coupled receptor gene that is homologous to the α₂-adrenoceptor. The cloned genomic DNA had no intron and specified an open reading frame of 1137 base pairs encoding 379 amino acids (aa). The predicted aa sequence has a typical seven hydrophobic transmembrane spanning region and a consensus G protein-binding motif. This receptor was most homologous to the human 5HT_1A receptor and closely related to other 5HT₁ receptor subtypes.     

The metamorphosis of visual systems in the sea lamprey

The life cycle of the sea lamprey, Petromyzon marinus, includes two metamorphoses. At the end of a period spent as a blind larva, buried in the mud of streams, a first metamorphosis prepares it to migrate downstream to the sea or a lake for its growth phase. Then, following a second metamorphosis, it migrates upstream as a sexually mature adult to spawn and die. The downstream migrants have a visual system based upon rhodopsin and vitamin A₁, whereas that of the upstream migrants is based upon porphyropsin and vitamin A₂. The livers contain vitamin A₁ at all stages. The sea lamprey therefore exhibits a metamorphosis of visual systems, like those observed earlier among amphibia. The presence of porphyropsin in this member of the most primitive living group of vertebrates, as in fishes and amphibia, supports the notion that porphyropsin may have been the primitive vertebrate visual pigment. Its association with fresh water existence throughout this range of organisms also is consistent with the view that the vertebrate stock originated in fresh water. The observation that in the life cycle of the lamprey rhodopsin precedes porphyropsin is not at variance with the idea that porphyropsin is the more primitive pigment, since this change is part of the second metamorphosis, marking the return to the original environment. The observation that in lampreys, fishes, and amphibia, porphyropsin maintains the same general association with fresh water, and rhodopsin with marine and terrestrial habit, suggests that a single genetic mechanism may govern this association throughout this wide span of organisms.     

Isolation, sequence analysis, and intron-exon arrangement of the gene encoding bovine rhodopsin

We have isolated cDNA clones generated from the mRNA encoding the opsin apoprotein of bovine rhodopsin and used these cDNAs to isolate genomic DNA clones containing the complete opsin gene. Nucleotide sequence analysis of the cloned DNAs has yielded a complete amino acid sequence for bovine rhodopsin and provided an intron-exon map of its gene. The mRNA homologous sequences in the 6.4 kb gene consist of a 96 bp 5' untranslated region, a 1044 bp coding region, and a surprisingly long approximately 1400 bp 3' untranslated region, and are divided into five exons by four introns that interrupt the coding region. Secondary structure analysis predicts that the bovine rhodopsin chain, like that of bacteriorhodopsin, contains seven transmembrane segments. Interestingly, three of the four introns are immediately distal to the codons for three of these segments, and one of these introns marks the boundary between the C-terminal domain and a transmembrane domain.     

Cloning of the cDNA and Genes for the Hamster and Human β2-Adrenergic Receptors

Abstract

The adenylate cyclase-stimulatory β₂-adrenergic receptor has been purified to apparent homogeneity from hamster lung. Partial amino acid sequence obtained from isolated CNBr peptides was used to clone the gene and cDNA for this receptor. The predicted amino acid sequence for the hamster β₂-adrenergic receptor revealed that the protein consists of a single polypeptide chain of 418 aa with consensus N-glycosylation and phosphorylation sites predicted by previous in vitro data. The most striking feature of the receptor protein however, is that it contains seven stretches of hydrophobic residues similar to the proposed seven transmembrane segments of the light receptor rhodopsin. Significant amino acid homology (30-35%) can be found between the hamster β₂-adrenergic receptor and rhodopsin within these putative membrane spanning regions. Using a hamster β₂-adrenergic receptor probe, the gene and cDNA for the human β₂-adrenergic receptor were isolated, revealing a high degree of homology (87%) between the two proteins from different species. Unlike the genes encoding the family of opsin pigments, of which rhodopsin is a member, the genes encoding both hamster and human β₂-adrenergic receptors are devoid of introns in their coding as well as 5′ and 3′ untranslated nucleotide sequences. The cloning of the genes and the elucidation of the aa sequences for these G-protein coupled receptors should help to determine the structure-function as well as the evolutionary relationship of these proteins.     

Cloning and characterization of two processed pseudogenes and the cDNA for the murine U1 snRNP-specific protein C

Genes for the snRNP proteins U1-70K, U1-A, Sm-B′/B, Sm-D1 and Sm-E have been isolated from various metazoan species. The genes for Sm-D1 and Sm-E, which were isolated from a murine and human source respectively, appear to belong to a multigene family. It has been suggested that also for the mammalian U1-C protein such a multigene family exists. With the human U1-C cDNA as a probe, two genes containing sequences homologous to the probe sequence were isolated from a mouse genomic library. Simultaneously, a murine U1-C cDNA was isolated from a mouse cDNA library. This 0.74 kb cDNA contains an open reading frame (ORF) of 477 bp encoding a polypeptide of 159 amino acids (aa) which differs at only one position (position 65) from the human U1-C protein. One of the isolated U1-C genes contains an ORF as well and shares 92% nucleotide sequence identity with the mouse U1-C cDNA. The features of this gene, in particular the absence of introns, the acquisition of a 3′ poly(A) tail and flanking direct repeats, indicate that it represents a processed pseudogene. At the predicted aa sequence level, substitutions of conserved residues at functionally important positions are observed, strongly suggesting that expression of this gene would not lead to a functional polypeptide. The second U1-C gene appeared to be a pseudogene as well because it is also intronless and contains a frameshift mutation compared to the ORF in the mouse U1-C cDNA. The characterization of these two pseudogenes points to the existence of a U1-C multigene family in mice. Furthermore, comparison of aa sequences of the murine, human and Xenopus U1-C shows that the protein is highly conserved through evolution. Since the Xenopus U1-C differs from the two mammalian counterparts solely at a number of positions in the C-terminal region, it can be concluded that aa changes are less well tolerated in the N-terminal region of U1-C than in the rest of the protein.     

Cloning and expression of two autolysin genes, cwlU and cwlV, which are tandemly arranged on the chromosome of Bacillus polymyxa var. colistinus

The cwlV gene, which encodes Bacillus polymyxa var. colistinus autolysin was cloned and sequenced. cwlV comprises a 1497-bp ORF and encodes a polypeptide of 499 amino acid (aa) residues (M_r of 53,707 Da). The N-terminal sequence of the mature 23-kDa CwlV protein is NSXGKKVVVIDAGXGAKD(X, undetermined aa); this processed form corresponds to the C-terminal portion (183?aa, M_r of 20,050?Da) of the cwlV ORF. Sequencing of the flanking region revealed that another putative autolysin gene, cwlU, is located upstream of cwlV. cwlU encodes a polypeptide of 524 aa and its deduced sequence is 34.9% identical to the full-length sequence of CwlV. Downstream of cwlV, the genes for a deduced lipoprotein (OrfW), an endonuclease III homolog (Nth), a non-homologous OrfX, a glutathione peroxidase homolog (Gpx), and the N-terminal region of OrfZ containing a ATP/GTP-binding site motif were found. Northern blotting and primer-extension analyses revealed that cwlU is transcribed as a single cistron, but cwlV is transcribed with orfW. The unprocessed forms of CwlV and CwlU (VΔS and UΔS, respectively) and their predicted mature forms (Vcat and Ucat, respectively) were expressed in, and purified from, Escherichia coli. Enzyme analysis indicated that VΔS and Vcat exhibit low and high cell wall hydrolase activities toward B. polymyxa cell wall, respectively, but UΔS and Ucat exhibit almost no and low cell wall hydrolase activities, respectively.     

Nucleotide sequence of the Synechococcus sp. PCC7942 branching enzyme gene (glgB): expression in Bacillus subtilis

The nucleotide sequence of the Synechococcus sp. PCC7942 glgB gene has been determined. The gene contains a single open reading frame (ORF) of 2322 bp encoding a polypeptide of 774 amino acids (aa) with an Mr of 89,206. Extensive sequence similarity exists between the deduced aa sequence of the Synechococcus sp. glgB gene product and that of the Escherichia coli branching enzyme in the middle portions of the proteins (62% identical aa). In contrast, the N-terminal portions shared little homology. The sequenced region which follows glgB contains an ORF encoding 79 aa of the N terminus of a polypeptide that shares extensive sequence similarity (41% identical aa) with human and rat uroporphyrinogen decarboxylase. This suggests that the region downstream from glgB contains the hemE gene and, therefore, that the organization of genes involved in glycogen biosynthesis in Synechococcus sp. is different from that described for E. coli. A fusion gene was constructed between the 5' end of the Bacillus licheniformis penP gene and the Synechococcus sp. glgB gene. The fusion gene was efficiently expressed in the Gram+ micro-organism Bacillus subtilis and specified a branching enzyme with an optimal temperature for activity similar to the wild-type enzyme.     

Isolation and sequence analysis of Clpgl,a gene coding for an endopolygalacturonase of the phytopathogenic fungus Colletotrichum lindemuthianum

Oligodeoxyribonucleotide primers designed from the N-terminal amino acid (aa) sequence of the endopolygalacturonase (EndoPG) of Colletotrichum lindemuthianum (Cl) race β and from an internal sequence conserved among different fungal EndoPG were used in a polymerase chain reaction (PCR) to amplify genomic related sequences of the fungus. A 542-bp fragment, designated pgA, was obtained and used as a probe to screen a partial genomic library of Cl. Among the positive clones, one was further analyzed. Nucleotide sequencing of this clone revealed an ORF encoding a 363-amino-acid (aa) polypeptide begining with a signal peptide of 26 aa interrupted by an intron of 70 bp, and showing a high degree of homology to ten fungal EndoPG sequences. Consensus sequences were identified in the 5′ non-coding region. This genomic clone was thereafter designated Clpgl. Southern analysis, performed with a Clpgl-specific probe, showed that this gene is present as a single copy in the Cl genome     

Cloning and characterization of the actin-encoding gene of Chlamydomonas reinhardtii

The genomic and complementary DNA sequences were determined for the unique actin-encoding gene in Chlamydomonas reinhardtii (Cr). The deduced amino acid (aa) sequence of this actin was similar to most known actin sequences, with the highest identity (98.1%) being with that of Volvox carteri actin. The Cr actin-encoding gene has one intron in the 5′-untranslated region and eight introns in the coding region. The latter eight introns occur at the same positions as those in the V. carteri actin-encoding gene. The 5′-upstream region contains four short stretches of sequence similar to the so-called ‘tub box’, a characteristic sequence proposed to be responsible for the regulation of synthesis of various axonemal proteins upon deflagellation and during the cell cycle. Southern blot analysis indicated that the Cr genome has only a single actin-encoding gene. An antibody specific for the 11-aa peptide corresponding to the N-terminal sequence of this actin was found to react with a 43-kDa protein associated with flagellar inner-arm dynein. These findings indicate that a single actin functions in both the cytoplasm and flagella of this organism.