A gene, SMP2, involved in plasmid maintenance and respiration in Saccharomyces cerevisiae encodes a highly charged protein

Summary The smp2 mutant of Saccharomyces cerevisiae shows increased stability of the heterologous plasmid pSR1 and YRp plasmids. A DNA fragment bearing the SMP2 gene was cloned by its ability to complement the slow growth of the smp2 smp3 double mutant (smp3 is another mutation conferring increased stability of plasmid pSR1). The nucleotide sequence of SMP2 indicated that it encodes a highly charged 95 kDa protein. Disruption of the genomic SMP2 gene resulted in a respiration-deficient phenotype, although the cells retained mitochondrial DNA, and showed increased stability of pSR1 like the original smp2 mutant. The fact that the smp2 mutant is not always respiration deficient and shows increased pSR1 stability even in a rho ⁰ strain lacking mitochondrial DNA suggested that the function of the Smp2 protein in plasmid maintenance is independent of respiration. The SMP2 locus was mapped at a site 71 cM from lys7 and 21 cM from ilv2/SMR1 on the right arm of chromosome XIII.     

Molecular cloning of three cDNAs encoding putative larval cuticle protein expressed differentially after larval ecdysis from the mulberry longicorn beetle, Apriona germari

Three cDNAs encoding putative larval cuticle protein (LCP) were cloned from the mulberry longicorn beetle, Apriona germari. The three cDNA sequences were 309 bp, 396 bp and 408 bp in length, encoding 103, 132 and 136 amino acid residues, respectively. The predicted molecular masses for these LCPs were approximately 9.2 kDa (AgLCP9.2), 12.3 kDa (AgLCP12.3) and 12.6 kDa (AgLCP12.6). Pairwise identity among AgLCP9.2, AgLCP12.3 and AgLCP12.6 were relatively low. Each AgLCP contained a type-specific consensus sequence identifiable in other insect cuticle proteins. The deduced amino acid sequence of AgLCP9.2 is most similar to Bombyx mori LCP18 and those of AgLCP12.3 and AgLCP12.6 are both most similar to B. mori LCP17. Northern blot analysis revealed that the three AgLCPs showed epidermis-specific expression. The expression profile of AgLCPs after larval ecdysis revealed by Northern blot analysis that the high-level mRNA expression of AgLCPs was detected on the first day of larval ecdysis for AgLCP9.2, on the fifth day for AgLCP12.3 and from the first day of larval ecdysis to the fifth day after larval ecdysis for AgLCP12.6, demonstrating that AgLCP mRNAs are differentially expressed in epidermis after larval ecdysis.     

Unusual sequence of an abscisic acid-inducible mRNA which accumulates late in Brassica napus seed development

We have analyzed the nucleotide sequence and accumulation of an mRNA which is prevalent in seeds of Brassica napus L. During normal development, the mRNA begins to accumulate during late embryogeny, is stored in dry seeds, and becomes undetectable in seedlings within 24 hours after imbibition. Moreover, abscisic acid treatment of embryos precociously induces or enhances accumulation of the mRNA. Nucleotide sequencing studies show that the deduced 30 kDa polypeptide has an unusual primary structure; the polypeptide possesses direct amino acid sequence repeats and is virtually entirely hydrophilic with the exception of a hydrophobic carboxyl-terminal region. Based upon the expression pattern and predicted polypeptide sequence, we conclude that the mRNA is encoded by a late embryogenesis-abundant (Lea) gene in B. napus.Abbreviations ABA abscisic acid - bp base pairs - DAF days after flowering - HAI hours after the start of imbibition - kb kilobase (pairs)     

Cloning and characterization of a novel human TEKTIN1 gene

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. A new member of the tektin gene family was cloned from the human fetal brain cDNA library. We hence named it the human TEKTIN1 gene. TEKTIN1 cDNA consists of 1375 bp and has a putative open reading frame encoding 418 amino acids. The predicted protein is 48.3 kDa in size, and its amino acid sequence is 82% identical to that of the mouse, rat, and dog. One conserved peptide RPNVELCRD was observed at position number 323–331 of the amino acid sequence, which is a prominent feature of tektins and is likely to represent a functionally important protein domain. TEKTIN1 gene was mapped to the human chromosome 17 by BLAST search, and at least eight exons were found. Northern blot analysis indicated that TEKTIN1 was predominantly expressed in testis. By in-situ hybridization analysis, TEKTIN1 mRNA was localized to spermatocytes and round spermatids in the seminiferous tubules of the mouse testis, indicating that it may play a role in spermatogenesis.     

Cloning and characterization of cDNAs coding for Vicia faba polyphenol oxidase

Three cDNA clones were isolated which code for the ubiquitous chloroplast enzyme, polyphenol oxidase (PPO), from Vicia faba. Analysis of the cloned DNA reveals that PPO is synthesized with an N-terminal extension of 92 amino acid residues, presumed to be a transit peptide. The mature protein is predicted to have a molecular mass of 58 kDa which is in close agreement to the molecular mass estimated for the in vivo protein upon SDS-PAGE. Differences in the DNA sequence of two full-length and one partial cDNA clones indicate that PPO is encoded by a gene family. Analysis of the deduced amino acid sequence shows that the chloroplast PPO shares homology with the 59 kDa PPOs in glandular trichomes of solanaceous species. A high degree of sequence conservation was found with the copper-binding domains of the 59 kDa tomato PPO as well as hemocyanins and tyrosinases from a wide diversity of taxa.     

Purification and characterization of homodimeric methylmalonyl-CoA mutase from<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

High activity (>60 munit/mg protein) of 5-deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase (EC 5.4.99.2) was constantly found during growth of a strain of the root-nodule-forming bacterium Sinorhizobium meliloti harboring an extra plasmid-encoded copy of the methylmalonyl-CoA-mutase-encoding bhbA gene. The enzyme was purified to homogeneity and characterized. The purified enzyme was found to be a colorless apo-form. The apparent molecular weight of the enzyme was calculated to be 165,000±5,000 by Superdex 200 HR gel filtration. SDS-PAGE of the purified enzyme resolved one protein band with an apparent molecular mass of 80.0±2.0 kDa, indicating that the S. meliloti enzyme is composed of two identical subunits. The NH₂-terminal sequence was identical to that predicted from the bhbA nucleotide sequence. Monovalent cations were required for enzyme activity.Abbreviations AdoCbl 5-Deoxyadenosylcobalamin - KPB Potassium phosphate buffer - MCM Methylmalonyl-CoA mutase - PVDF Polyvinylidene difluoride     

A barley cDNA clone encoding a type III chlorophyll a/b-binding polypeptide of the light-harvesting complex II

The nucleotide sequence of a leaf cDNA clone encoding a Type III chlorophyll a/b-binding (CAB) protein of light-harvesting complex II (LHCII) in barley is reported. Sequence comparisons and results from in vitro import into chloroplasts demonstrate that the cDNA clone encodes a functional transit peptide of 45 amino acid residues and a mature polypeptide of 223 residues with a predicted molecular mass of 24.3 kDa. After insertion into thylakoids, the mature protein is resistant to protease attack. Hybridization analysis using a gene-specific probe shows that the gene is expressed in dark-grown seedlings and that the amount of mRNA increases during illumination.     

Identification of a Novel mRNA-Associated Protein in Oocytes ofPleurodeles waltlandXenopus laevis

Amphibian oocytes accumulate a large pool of mRNA molecules for future embryonic development. Due to their association with specific proteins the stored maternal RNAs are translationally repressed. The identification of these RNA-binding proteins and the characterization of their functional domains may contribute to the understanding of the translational repression mechanisms and the subsequent activation processes during early embryogenesis. Here we present the completePleurodelescDNA sequence of a cytoplasmic protein which is present in oocytes, eggs, and very early cleavage stage embryos but undetectable in postcleavage embryo and adult tissues. The predicted molecular mass of the protein is 55 kDa and the apparent molecular mass as determined by SDS–PAGE, 68 kDa. The deduced amino acid sequence reveals proline- and serine-rich domains in the aminoterminal part as well as two RGG boxes which represent characteristic motifs of several RNA-binding proteins. No distinct homologies to the consensus RNA recognition motif were found. The 55-kDa protein was recovered in cytoplasmic ribonucleoprotein (RNP) particles containing poly(A)⁺RNA. It was therefore termed RAP55 for mRNA-associated protein of 55 kDa. However, a direct interaction of RAP55 with mRNA could not be demonstrated by UV-crosslinking experiments, indicating that it is bound to mRNP complexes via protein–protein interactions. RAP55 is evolutionarily conserved since antibodies raised against a recombinantPleurodelesRAP55 fragment recognize the protein fromPleurodelesandXenopus.The expression pattern and intracellular distribution of RAP55 suggest that it is part of those mRNP particles which are translationally repressed during oogenesis and become activated upon progesterone-induced oocyte maturation.     

Identification and Characterization of Putative Receptors for Sperm-Activating Peptide I (SAP-I) in Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus1

We characterized putative receptors specific for sperm-activating peptide I (SAP-I: GFDLNGGGVG) in spermatozoa of the sea urchin Hemicentrotus pulcherrimus, using both binding and crosslinking techniques. Analysis of the data obtained from the equilibrium binding of a radioiodinated SAP-I analogue [GGGY(¹²⁵I)-SAP-I] to H. pulcherrimus spermatozoa showed the presence of two classes of receptors specific for SAP-I in the spermatozoa. The incubation of intact spermatozoa as well as sperm tails or sperm membranes prepared from H. pulcherrimus spermatozoa with GGGY(¹²⁵I)-SAP-I and a chemical crosslinking reagent, disuccinimidyl suberate, resulted in the radiolabelling of a 71 kDa protein. The protein appears to be associated with a 220 kDa wheat germ agglutinin (WGA)-binding protein. A cDNA encoding the 71 kDa protein was isolated from a H. pulcherrimus testis cDNA library. The cDNA was 2443 bp long and an open reading frame predicted a protein of 532 amino acids containing a 30-residue amino-terminal signal peptide, followed by the same sequence as the N-terminal sequence of the 71 kDa protein. The amino acid sequence of the matured 71 kDa protein is strikingly similar to the 77 kDa protein of Strongylocentrotus purpuratus (95.5% identical) and also similar to cysteine rich domain of a human macrophage scavenger receptor. Northern blot analysis demonstrated that mRNA of 2.6 kb encoding the 71 kDa protein was expressed only in the testis.     

β-Defensin from the Asian Sea Bass,Lates calcarifer: Molecular Prediction and Phylogenetic Analysis

Antimicrobial peptides (AMPs) are an important element of the innate immune system of all living organisms and serve as a barrier that safeguards the organisms against a wide range of pathogens. Fishes are proven to be a prospective source of AMPs, and β-defensins form an important family of AMPs with potent antimicrobial, chemotactic and immunomodulatory activities. The present study reports a β-defensin AMP sequence (Lc-BD) from the Asian sea bass, Lates calcarifer, a commercially important fish species in tropical and subtropical regions of Asia and the Pacific. A 202-bp cDNA fragment with an open reading frame encoding 63 amino acids (aa) was obtained from the mRNA of gill tissue by RT-PCR. The deduced aa sequence of Lc-BD possessed a signal and a mature peptide region with 20 and 43 aa residues, respectively. Lc-BD was characterized at the molecular level, and a molecular weight of 5.24 kDa and a net charge of +4.5 was predicted for the mature peptide. The molecular characterization of Lc-BD revealed the presence of three intramolecular disulphide bonds involving the six conserved cysteine residues in the sequence, and the phylogenetic analysis of Lc-BD showed a close relationship with β-defensins from fishes like Siniperca chuatsi, Argyrosomus regius, Trachinotus ovatus and Oplegnathus fasciatus.

     

Cloning and molecular analysis of the bifunctional dihydrofolate reductase-thymidylate synthase gene in the ciliated protozoanParamecium tetraurelia

We have cloned the first bifunctional gene dihydrofolate reductase-thymidylate synthase (DHFR-TS) from a free-living, ciliated protozoan,Paramecium tetraurelia, and determined its macronuclear sequence using a modified ligation-mediated polymerase chain reaction (PCR) that can be of general use in cloning strategies, especially where cDNA libraries are limiting. While bifunctional enzyme sequences are known from parasitic protozoa, none had previously been found in free-living protozoa. The AT-rich (68%) coding region spanning 1386 bp appears to lack introns. DHFR-TS localizes to a 500 kb macronuclear chromosome and is transcribed as an mRNA of 1.66 kb, predicted to encode a 53 kDa protein of 462 residues. The N-terminal one-third of the protein is encoded by DHFR, which is joined by a short junctional peptide of 12 amino acids to the highly conserved C-terminal TS domain. Among known DHFR-TS sequences, theP. tetraurelia gene is most similar to that fromToxoplasma gondii, based on primary sequence and parsimony analyses. The predicted secondary protein structure is similar to those of previously crystallized monofunctional sequences.     

A putative catabolite-repressed cell wall protein from the mycoparasitic fungus Trichoderma harzianum

A cDNA clone encoding a putative cell wall protein (Qid3) was isolated from a library prepared from chitin-induced mRNA in cultures of the mycoparasitic fungus Trichoderma harzianum. The predicted 14 kDa protein shows a potential signal peptide, several hydrophobic domains and certain motifs that are structurally similar to proline-rich and glycine-rich plant cell wall proteins. Expression of the qid3 gene is derepressed in the absence of glucose. When introduced in yeast, qid3 expression causes cell division arrest into cytokinesis and cell separation, probably due to its cell wall localization.     

Isolation and characterization of two wound-regulated tomato extensin genes

Extensins comprise a family of structural cell wall hydroxyproline-rich glycoproteins in plants. Two tomato genomic clones, Tom J-10 and Tom L-4, were isolated from a tomato genomic DNA library byin situ plaque hybridization with extensin DNA probes. Tom J-10 encoded an extensin with 388 amino acid residues and a predicted molecular mass of 43 kDa. The Tom J-10 encoded extensin lacked a typical signal peptide sequence, but contained two distinct protein domains consisting of 19 tandem repeats of Ser-Pro₄-Ser-Pro-Lys-Tyr-Val-Tyr-Lys at the amino terminus which were directly followed by 8 tandem repeats of the consensus sequence Ser-Pro₄-Tyr₃-Lys-Ser-Pro₄-Ser-Pro at the carboxy terminus. RNA blot hybridization analysis with the Tom J-10 extensin probe demonstrated the presence of a 4.0 kb tomato stem mRNA which accumulated markedly in response to wounding. Tom L-4 encoded an extensin with 322 amino acid residues and a predicted molecular mass of 35 kDa. The Tom L-4 encoded extensin contained a typical signal peptide sequence at the amino terminus and was followed by at least 3 distinct domains. These domains consisted of an amino terminal domain containing several Lys-Pro and Ser-Pro₄ repeat units, a central domain with repeats of the consensus sequence Ser-Pro_2–5-Thr-Pro-Ser-Tyr-Glu-His-Pro-Lys-Thr-Pro, and a carboxy terminal domain containing repeats of the consensus sequence Ser-Ser-Pro₄-Ser-Pro-Ser-Pro₄-Thr-Tyr_1–3. RNA blot hybridization analysis with the Tom L-4 extensin probe demonstrated the presence of a 2.6 kb tomato stem mRNA which accumulated in response to wounding.     

Mutations in a Saccharomyces cerevisme host showing increased holding stability of the heterologous plasmid pSRI

Summary We have isolated Saccharomyces cerevisiae mutants, smp, showing stable maintenance of plasmid pSRI, a Zygosaccharomyces rouxii plasmid. The smp mutants were recessive and were classified into at least three different complementation groups. The three mutants also showed increased stability of YRp plasmids and the mutations are additive for plasmid stability. One mutation, smp1, confers a respiration-deficient (rho ⁰) phenotype and several Rho^– mutants independently isolated by ethidium bromide treatment of the same yeast strain also showed increased stabilities of pSR1 and YRp plasmids. The wild-type S. cerevisiae cells showed a strongly biased distribution of pSR1 molecules as well as YRp plasmids to the mother cells at mitosis, while the smpf mutant did not show this bias. Another mutation, smp3, at a locus linked to ade2 on chromosome XV, confers temperature-sensitive growth. The SMP3 gene encodes a 59.9 kDa hydrophobic protein and disruption of the gene is lethal.     

Sequence structure and expression pattern of a novel anionic defensin-like gene from silkworm (Bombyx mori)

A defensin-like gene, BmdefA, was rediscovered in the silkworm genome and expressed sequence tags databases. The open reading frame of BmdefA encodes a prepropeptide consisting of a 22-residue signal peptide, a 34-residue propeptide, and a 36-residue mature peptide with a molecular mass of 4.0 kDa. The mature peptide possesses the characteristic six-cysteine motif of insect defensins, and its predicted isoelectric point is 4.12, indicating it is a novel anionic defensin. An intron is present in BmdefA and several cis-regulatory elements are in the regulating region. It is transcribed constitutively at a high level in the hemocyte, silk gland, head, and ovary of the silkworm larvae, and in the fat body of early-stage pupae and moth. BmdefA is also strongly induced by immune challenge. These results suggest that BmdefA plays an important role in both immunity and metamorphosis. Hongxiu Wen and Xiqian Lan contributed equally to this work and should be considered co-first authors.     

Expression of an AT-rich xylanase gene from the anaerobic fungus <Emphasis Type="Italic">Orpinomyces</Emphasis> sp. strain PC-2 in and secretion of the heterologous enzyme by <Emphasis Type="Italic">Hypocrea jecorina</Emphasis>

The catalytic domain encoded by an adenine–thymine (AT)-rich xylanase gene (xynA) of the anaerobic fungus Orpinomyces was expressed in Hypocrea jecorina under the control of the cel7A promoter and terminator. No XynA protein was detected in H. jecorina culture supernatants when the original sequence was fused to the H. jecorina cel5A region coding for its signal peptide, carbohydrate-binding module, and hinge. Replacing the xynA (56% AT content) with a synthetic sequence containing lower AT content (39%) supported the extracellular production (150 mg l⁻¹) of the fusion xylanase by H. jecorina. Northern analysis revealed that successful production after the decrease in AT content was related to higher levels of the xylanase-specific mRNA. Another construct with an RDKR-coding sequence inserted between the cel5A linker and the xynA catalytic domain allowed production of the fully processed active xylanase catalytic domain. Both the fusion (40 kDa) and the fully processed (28 kDa) forms displayed enzymatic properties of family 11 xylanases. Both the R and the Kex2-like KR sites were recognized during secretion, resulting in a mixture of two amino termini for the 28-kDa xylanase. The work demonstrated for the first time that glycoside hydrolases derived from anaerobic fungi can be produced by H. jecorina. The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

The virB operon of Agrobacterium tumefaciens pTiC58 encodes 11 open reading frames

Summary Agrobacterium tumefaciens genetically transforms plant cells by transferring a copy of its T-DNA to the plant where it is integrated and stably maintained. In the presence of wounded plant cells this process is activated and mediated by the products of the vir genes which are grouped into six distinct loci. The largest is the virB locus spanning 9.5 kb. Transposon mutagenesis studies have shown that virB gene products are required for virulence but their functions remain largely unknown. To provide information relevant to understanding the function of VirB polypeptides, the nucleotide sequence of the virB operon from a nopaline plasmid, pTiC58, is presented here. Eleven open reading frames (ORFs) are predicted from this sequence. The predicted sizes of 10 of the 11 VirB polypeptides are verified by specific expression in Escherichia coli. Only the product of the smallest ORF potentially encoding a 5.8 kDa polypeptide has not been detected. The initiation of translation of five virB ORFs occurs at codons that overlap the termination codons of the ORF immediately upstream; thus, translational coupling may be an important mechanism for efficient translation of the large virB polycistronic mRNA. Based on hydropathy plot analysis nine of the virB ORFs encode proteins that may interact with membranes; these data support the earlier hypothesis (Engstromm et al. 1987) that virB gene products may form a membrane pore or channel to mediate exit of the T-DNA copy (T-strands) from Agrobacterium into the plant cell. A comparison of the two published octopine virB sequences with the nopaline sequence presented here is made.     

Extracellular metalloendopeptidase of Streptomyces rimosus

Metalloendopeptidase was isolated from Streptomyces rimosus culture filtrates in a homogeneous form. It was determined to be a 15 kDa basic protein, most active around pH 7.5, and susceptible to inhibition by chelating agents, N-bromosuccinimide, thiorphan, and 10⁻⁴ M zinc. The enzyme was highly specific for phenylalanine at the N-side of endopeptide bonds. Determination of amino acid sequence of the enzyme’s NH₂-part allowed the recognition of its structure homology with isolated and predicted metallopeptidases from several Streptomyces species. The data contribute to the definition of M7 family of metalloendopeptidases in streptomycetes.