A novel response-regulator is able to suppress the nodulation defect of a Bradyrhizobium japonicum nodW mutant

The two-component regulatory system Nod-VW of Bradyrhizobium japonicum is essential for the nodulation of the legume host plants Vigna radiata, V. unguiculata and Macroptilium atropurpureum. The NodV protein shares homology with the sensor-kinases, whereas the NodW protein is a member of the response-regulator class. We report here the identification of a new B. japonicum DNA region that is able to suppress the phenotypic defect of a nodW mutant, provided that this region is expressed from a foreign promoter. The minimal complementing region, which itself is not essential for nodulation in a nodW ⁺ background, consists of one gene designated nwsB (nodW-suppressor). The deduced amino acid sequence of the nwsB gene product shows a high degree of homology to NodW. The nwsB gene is preceded by a long open reading frame, nwsA, whose putative product appears to be a sensor-kinase. Downstream of nwsB, an open reading frame encoding a second putative response-regulator was identified. Interspecies hybridization revealed the presence of nwsAB-like DNA also in other Bradyrhizobium strains. Using nwsB′-′lacZ fusions, the nwsB gene was found to be expressed rather weakly in B. japonicum. This low level of expression is obviously not sufficient to compensate for a nodW ^? defect, whereas strong overexpression of nwsB is a condition that leads to suppression of the nodW ^? mutation.     

Characterization of Geranium (Pelargonium graveolens) Chloroplast EF-Tu cDNA

A geranium (Pelargonium graveolens) chloroplast translational elongation factor EF-Tu (tufA) cDNA was isolated. The geranium tufA cDNA is 1,584 bp long with 20 bp of 5 untranslated region (UTR) and 139 bp of 3 UTR. It encodes 474 amino acids including a putative chloroplast transit peptide of 65 amino acids. The deduced polypeptides of the geranium tufA cDNA contains four GTP binding sequences in its N-terminal region and two chloroplast EF-Tu signature regions in the C-terminal region. The predicted molecular weight of the mature geranium chloroplast EF-Tu protein was about 45,000 and its amino acid sequence identity with the chloroplast EF-Tu proteins of tobacco, pea, Arabidopsis, rice, and soybean ranges from 85% to 91%. The geranium tufA appears to exist as a single copy gene like Arabidopsis and rice, whereas other known dicot plants have more than one copy in their nuclear genomes.     

Expression and analysis of the rat placental class I cDNA clone encoding the Pa antigen

The previously sequenced cDNA clone pARL 5 was recloned into the mammalian expression vector pcEXV3, and transient and permanent transfectants were prepared in COS7 green monkey kidney fibroblasts. The transfectants were analyzed by indirect immunofluorescence using monoclonal and polyclonal antibodies raised in specifically selected rat strain combinations. These studies showed that pARI.5 encodes the Pa antigen and that the Pa molecule is distinct from the A^a molecule. Probes were derived from the pARL5 clone and used to study the genomic DNA from Pa-positive and Pa-negative strains. Two probes derived from the 3' untranslated region (3apARI.5 and 3bpARI.5) and one probe derived from the 5' region (5pARI.5) hybridized nonspecifically in all strains under moderate stringency conditions. By contrast, anXba I restriction fragment unique to thePa gene was detected with the (5pARI.5) probe under high stringency conditions. This probe hybridized with a 1.8 kilobase (kb) fragment in the Pa-positive strains and with a 1.7 kb band in the Pa-negative strains. These studies suggest that the gene encoding the Pa antigen, or a fragment thereof, is present in both Pa-positive and Panegative strains but may not be expressed in the latter.     

Abundance and half-life of the distinct oat phytochrome A3 and A4 mRNAs

Gene-preferential oligonucleotide probes were used to determined the relative abundance and half-lives of distinct oat phytochrome A (PHYA) mRNAs. Oat PHYA mRNAs are highly conserved in the 5-untranslated region and the coding region, but the 3-untranslated region has an overall lower sequence conservation and was the source of gene-preferential probes. PHYA3 mRNA was estimated to be ca. 61% of the oat PHYA mRNA pool present in poly(A)⁺ RNA from dark-grown seedlings. The half-lives for PHYA3 and PHYA4 mRNAs were both estimated to be ca. 30 min, and a similar short half-life was estimated for the average PHYA mRNA. Sequence comparisons of PHYA mRNAs from four grass species identified conserved sequences within the 5- and 3-untranslated regions that might be important for PHYA mRNA degradation.     

UV-mutable hybrids of Salmonella incorporating escherichia coli Region adjacent to Tryptophan operon

Summary Salmonella typhimurium recombinants have been constructed which have acquired UV-induced mutability. Such recombinants were obtained after transfer of plasmid F126 from E. coli. UV-mutability was acquired by UV-stable Salmonella as a nonselective marker after selection of trp ⁺ marker in 2.5% of the cases. Possible deficiency in UV-stable Salmonella of the umuC gene is discussed.     

Structure and expression during the germination of rice seeds of the gene for a carboxypeptidase

The carboxypeptidase gene from rice and corresponding cDNA clones were isolated. The SalI 11.2 kb fragment of DNA cloned from a size-fractionated genome library contained eight introns and an open reading frame that encoded 500 amino acids (M _r 55445). The structure deduced for the carboxypeptidase from rice was very similar to those of type III serine carboxypeptidases from barley and wheat. The extent of homology of the amino acid sequence to that of these carboxypeptidases from barley and wheat was 92.3% and 87.2%, respectively. The accumulation of mRNA for the rice carboxypeptidase was conspicuous in germinating endosperms that contained aleurone layers, but levels were lower in leaves and roots. The abundance of the mRNA in endosperms was enhanced by gibberellic acid (GA) and accumulation of the mRNA was inhibited by abscisic acid (ABA). The rice gene for carboxypeptidase contained some pyrimidine boxes (_T ^CCTTTT_T ^C), in the 5 flanking region, which are a characteristic of a GA-responsive gene.     

DNA sequence of a class I pseudogene from theTla region of the murine MHC: Recombination at a B2 Alu repetitive sequence

Summary We have determined the DNA sequence of a BALB/cTla region class I gene from the major histocompatibility complex (MHC) that had been identified previously as encoding a murine antigen by DNA-mediated gene transfer. Analysis of the DNA sequence shows, however, that this gene, the T1^c gene from theTla ^c genotype, could not encode a TL antigen or any other functional class I molecule due to the presence of numerous stop codons and frameshift mutations in the coding regions. This result suggests that the earlier transformation data may have been incorrect or perhaps that the clone containing the T1^c gene contains sequences that induced expression of a serologically reactiveTla gene in the genome of the recipient L cell. The T1^c gene is structurally related to the previously sequenced T13^c gene that encodes a serologically defined TL antigen. The 3 half of the T1^c gene including exons 4, 5, 6, and the 3 untranslated region has about 85% nucleotide similarity (including introns) with the corresponding parts of the T13^c gene; however, the 5 half of the T1^c gene has little homology with the T13^c gene. There is a sharp line of demarcation between the homologous and nonhomologous regions, and this border occurs precisely at a B2 Alu repeat sequence present in the T13^c gene. This suggests that a recombination event took place here and that an Alu repeat sequence that is known to have characteristics of transposable elements played some role in a recombination or gene conversion event.     

A newly discovered tRNA(1Asp) gene (aspV) of Escherichia coli K12

Summary We report a new tRNA ₁ ^Asp gene near the dnaQ gene, which is located at 5 min on the Escherichia coli linkage map. We named it aspV. The sequence corresponding to the mature tRNA is identical with that of the two previously identified tRNA ₁ ^Asp genes (aspT and aspU), but there is no homology in the sequences of their 3-and 5-flanking regions.Abbreviations kb kilo base pair(s) - rrn ribosomal RNA     

Nucleotide sequence of the split tRNAUAALeu gene from Vicia faba chloroplasts: evidence for structural homologies of the chloroplast tRNALeu intron with the intron from the autosplicable Tetrahymena ribosomal RNA precursor

Summary The gene encoding the tRNA _UAA ^Leu from broad bean chloroplasts has been located on a 5.1 kbp long BamHI fragment by analysis of the DNA sequence of an XbaI subfragment. This gene is 536 bp long and is split in the anticodon region. The 451 bp long intron shows high sequence homology over about 100 bp from each end with the corresponding regions of the maize chloroplast tRNA _UAA ^Leu intron. These conserved sequences are probably involved in the splicing reaction, for they can be folded into a secondary structure which is very similar to the postulated structure of the intron from the autosplicable ribosomal RNA precursor of Tetrahymena. Very little sequence conservation is found in the 5-and 3-flanking regions of the broad bean and maize chloroplast tRNA _UAA ^Leu genes.     

Nucleotide sequence of the 16S-23S spacer region in the rrnA operon from a blue-green alga, Anacystis nidulans

Summary The nucleotide sequence of an entire spacer region between the 16S and 23S rRNA genes of the rrnA operon from a blue-green alga, Anacystis nidulans, has been determined. The spacer region is 545 base pairs long and encodes tRNA^fle and tRNA^Ala in the order of 16S rRNA-tRNA^fle-tRNA^Ala-23S rRNA. A striking feature is that the A. nidulans tRNA^fle gene contains no 3-CCA sequence while the tRNA^Ala gene does. These spacer tRNA genes show strong sequence homology with those of chloroplasts and bacteria.     

Sensitive 1H-31P correlations with 5′ methylene protons of DNA via homonuclear double-quantum coherence

A novel pulse sequence is presented for the correlation of 5 and 5 protons in DNA with phosphorus. Double-quantum coherence between the methylene protons is used to generate ¹H5-³¹P and ¹H5-³¹P cross peaks in an HMQC-type experiment. The resolution for these cross peaks is significantly improved over that of conventional HSQC experiments, as cross peaks between ¹H4 and ³¹P are largely suppressed and a 3D version of the experiment can be performed with little penalty in sensitivity. In addition, sensitivity is favoured by slower relaxation of the double-quantum coherence and a more favourable multiplet fine structure in the acquisition dimension.     

Genetic and biochemical studies on flavonoid 3′-hydroxylation in flowers of Petunia hybrida

Summary In flower extracts of defined genotypes of Petunia hybrida, an enzyme activity was demonstrated which catalyses the hydroxylation of naringenin and dihydrokaempferol in the 3-position. Similar to the flavonoid 3-hydroxylases of other plants, the enzyme activity was found to be localized in the microsomal fraction and the reaction required NADPH as cofactor. A strict correlation was found between 3-hydroxylase activity and the gene Ht1, which is known to be involved in the hydroxylation of flavonoids in the 3-position in Petunia. Thus, the introduction of the 3-hydroxyl group is clearly achieved by hydroxylation of C₁₅-intermediates, and the concomitant occurrence of the 3,4-hydroxylated flavonoids quercetin and cyanidin (paeonidin) in the presence of the functional allele Ht1 is due to the action of one specific hydroxylase catalysing the hydroxylation of common precursors for both flavonols and anthocyanins.     

Sequence of the t complex Tcp-10a t gene and examination of the Tcp-10 t gene family

Transmission ratio distortion (TRD) is a property of complete t haplotypes which results in the preferential transmission of the t haplotype chromosome from heterozygous t/+ males to the majority of the offspring. A candidate gene for one of the primary genetic elements in TRD, the t complex responder locus has recently been suggested to be Tcp-10b ^t. There are multiple, functional Tcp-10 ^t genes, but genetic data suggest the presence of the Tcp-10a ^t gene alone is compatible with normal transmission ratios. Here we present the complete sequence and genomic structure of the Tcp-10a ^t gene which is compared with sequence data from a number of cDNAs and genomic subclones representing all active Tcp-10 ^t family genes. A detailed table of all sequence variants discovered in the course of our investigation is presented, and we have clarified the extent of 5 untranslated alternative splicing patterns exhibited by this gene family. A 60 base pair (bp) in-frame deletion from the 5 end of exon 3 of the Tcp-10a ^t gene is also presented and compared with the equivalent region of Tcp-10b ^t and Tcp-10c ^t. A search of the University of Edinburgh database has revealed a significant homology between the Tcp-10b ^t open reading frame and several cytosolic filament proteins. Interestingly, the region of homology is involved in the deletion from the Tcp-10a ^t gene.     

Cloning and characterisation of the recA gene of Agrobacterium tumefaciens C58

Summary A gene library of Agrobacterium tumefaciens C58 has been constructed in the plasmid vector pACYC184. A recombinant plasmid was isolated from the library by interspecific complementation in E. coli, which contained the A. tumefaciens recA gene. Heterologous Southern blotting and DNA sequence analysis have demonstrated the existence of considerable homology between the recA genes of A. tumefaciens, E. coli and R. meliloti.Abbreviations MMS methyl methanesulfonate - UV ultraviolet light - bp base pairs - kbp kilo base pairs - dATP deoxyadenosine 5-triphosphate - dNTP deoxynucleoside triphosphate - Ap ampicillin - Cm chloramphenicol - Km kanamycin - Tet tetracycline     

Identification of a PstI polymorphism in the p21Cip1/Waf1 cyclin-dependent kinase inhibitor gene

A PstI polymorphism in the 3 flanking region of the p21^CiP1/Waf1 cyclin-dependent kinase inhibitor gene is described. DNA sequencing analysis identified a CT base substitution in the 3 flanking region of the gene. This substitution leads to the destruction of a PstI site and results in a biallelic DNA polymorphism. This restriction fragment length polymorphism (RFLP) provides the first known genetic marker for this cell cycle regulatory gene.     

Chemical semisynthesis of aprotinin homologues and derivatives mutated inp′ positions

An extended concept for the replacement of amino acids in theP' region of aprotinin by chemical semisynthesis is presented. Either fragment condensation with dipeptides protected as tert-butyl ester or stepwise introduction of two single amino acid-tert-butyl esters into a partially esterified aprotinin derivative (with free Lys¹⁵-carboxyl group) lacking the amino acids Ala¹⁶ and Arg¹⁷ leads to aprotinin homologues and derivatives mutated in theP ₁ andP ₂ position. This method may complement the recently reported enzymatic synthesis by enabling access to aprotinin homologues and derivatives, which cannot be prepared enzymatically. The synthesis of [Ala¹⁷]BPTI and [seco-17/18]BPTI is described in detail.     

Cloning and characterization of a novel NK cell-specific serine protease gene and its functional 5′-flanking sequences

Rat natural killer cell Met-ase-1 (RNK-Met-1) is a 30 000 M _r serine protease (granzyme) found in the cytolytic granules of CD3- large granular lymphocytes (LGL) with natural killer (NK) activity. To characterize the genomic sequences responsible for the CD3- LGL-restricted expression of this gene, we screened a rat genomic library with RNK-Met-1 cDNA, and obtained bacteriophage clones that contained the RNK-Met-1 gene. The RNK-Met-1 gene comprises 5 exons and spans approximately 5.2 kilobases (kb), exhibiting a similar structural organization to a class of CTL-serine proteases with protease catalytic residues encoded near the borders of exons 2, 3, and 5. The 5-flanking region of the RNK-Met-1 gene contains a number of putative promoter and enhancer regulatory elements and shares several regions of homology with the 5-flanking region of the mouse perforin gene. We have prepared nested deletions from approximately 3.3 kb of the 5-flanking region of the RNK-Met-1 gene, and inserted these upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These 5-flanking RNK-Met-1-CAT constructs were transiently transfected into rat LGL leukemia, T-lymphoma, and basophilic leukemia cell lines.The nucleotide sequence data reported in this Papershave been submitted to the EMBL/GenBank nucleotide sequence database and have been assigned the accession number L38482.     

Geometry of the dry-state oligomerization of 2′,3′-cyclic phosphates

Summary Evaporation of a solution of thymidine plus either theexo or theendo diastereomer of uridine cyclic 2,3-O, O-phosphorothioate (U > p(S) in 1,2-diaminoethane hydrochloride buffer gave the 2,5 and 3,5 isomers of (P-thio) uridylylthymidine (Up(S)dT) in a ratio of 1:2 with a combined yield of about 20%. These isomers were re-converted to U > p(S) and dT by a reaction that is known to proceed by an in-line mechanism. Both the 2,5 and 3,5 isomers gave as product the same diastereomer of U > p(S) that had been used originally in their formation. These dry-state prebiotic reactions (Verlander, Lohrmann, and Orgel 1973) are thus shown to be stereospecific, and both the 2,5 and 3,5 internucleotide bonds are formed by an in-line mechanism.Abbreviations DAE 1,2-diaminoethane - HPLC high pressure liquid chromatography - RNase bovine pancreatic ribonuclease A, EC 3.1.4.22 - TEAB triethylammonium bicarbonate - tris tris(hydroxymethyl)aminomethane - UMP(S) uridine monophosphorothioate - U > p uridine cyclic 2,3-phosphate - U > p(S) uridine cyclic 2,3-O, O-phosphorothioate - Up(S)dT (P-thio)uridylylthymidine - U2p(R_p-S)5dT (P-thio)uridylylthymidine with theR configuration at phosphorous, and a 2,5 internucleotide linkage