On immunoglobulin heavy chain gene switching: two gamma 2b genes are rearranged via switch sequences in MPC-11 cells but only one is expressed

During B lymphocytes differentiation, switches in the expression of heavy chain immunoglobulin constant region (CH) genes occur by a novel DNA recombination mechanism. We have investigated the requirements of the CH gene switch by characterizing two rearranged gamma 2b genes from a gamma 2b producing mouse myeloma (MPC-11). One of the two gamma 2b genes is present in 2-3 copies per cell (gamma 2b strong hybridizer) while the other is present in approximately 1 copy per cell (gamma 2b weak hybridizer). Genomic clones of the gamma 2b strongly hybridizing gene indicate that this is an abortive switch event between the S gamma 3 and S gamma 2b regions. However, clones of the gamma 2b weakly hybridizing gene suggest a functional rearrangement due to the presence of VH, JH and S mu sequences. The switch-recombination sites of these rearranged gamma 2b genes and those of other CH genes show a high degree of preference for the sequence AGGTTG 5' of either the S mu donor site or the appropriate CH S acceptor site. AGGTTG and its analogs are rare in the S mu region, are somewhat prevalent in s alpha and in the case of S mu are found 5' of a tandemly repeated DNA sequence (GAGCT, GGGGT) comprising most of S mu.     

Chloroplast and cytosolic glutamine synthetase are encoded by homologous nuclear genes which are differentially expressed in vivo

We have shown that the individual members of the plant gene family for glutamine synthetase (GS) are differentially expressed in vivo, and each encode distinct GS polypeptides which are targeted to different subcellular compartments (chloroplast or cytosol). At the polypeptide level, chloroplast GS (GS2) and cytosolic GS (GS1 and GSn) are distinct and show an organ-specific distribution. We have characterized full length cDNA clones encoding chloroplast or cytosolic GS of pea. In vitro translation products encoded by three different GS cDNA clones, correspond to the mature GS2, GS1, and GSn polypeptides present in vivo. pGS185 encodes a precursor to the chloroplast GS2 polypeptide as shown by in vitro chloroplast uptake experiments. The pGS185 translation product is imported into the chloroplast stroma and processed to a polypeptide which corresponds in size and charge to that of mature chloroplast stromal GS2 (44 kDa). The 49 amino terminal amino acids encoded by pGS185 are designated as a chloroplast transit peptide by functionality in vitro, and amino acid homology to other transit peptides. The cytosolic forms of GS (GS1 and GSn) are encoded by highly homologous but distinct mRNAs. pGS299 encodes the cytosolic GS1 polypeptide (38 kDa), while pGS341 (Tingey, S. V., Walker, E. L., and Coruzzi, G. M. (1987) EMBO. J. 6, 1-9) encodes a cytosolic GSn polypeptide (37 kDa). The homologous nuclear genes for chloroplast and cytosolic GS show different patterns of expression in vivo. GS2 expression in leaves is modulated by light, at the level of steady state mRNA and protein, while the expression of cytosolic GS is unaffected by light. The light-induced expression of GS2 is due at least in part to a phytochrome mediated response. Nucleotide sequence analysis indicates that chloroplast and cytosolic GS have evolved from a common ancestor and suggest a molecular mechanism for chloroplast evolution.     

Tryptophan decarboxylase is encoded by two autonomously regulated genes in Camptotheca acuminata which are differentially expressed during development and stress

Camptothecin (CPT) is a valuable anti-cancer monoterpene alkaloid produced by the Chinese tree Camptotheca acuminata . Tryptophan decarboxylase (TDC) supplies tryptamine for the indole moiety of CPT and its derivatives, and is considered a key step in monoterpene indole alkaloid biosynthesis as it links primary and secondary metabolism. This report describes the isolation and characterization of tdc1 and tdc2 , two autonomously regulated TDC genes from Camptotheca . When expressed in Escherichia coli , the products of each gene could decarboxylate tryptophan, but were inactive against tyrosine, phenylalanine and 3,4-dihydroxyphenylalanine (dopa). tdc1 was developmentally regulated, having its highest expression level in the apex, young stem and bark, tissues which also contain the highest levels of CPT. Expression of tdc1 also increased during seedling development and was correlated with alkaloid accumulation during germination. tdc2 expression was induced in Camptotheca leaf discs and cell suspension cultures treated with fungal elicitor or methyl jasmonate, treatments which did not affect tdc1 expression. Unlike tdc1 , tdc2 expression was not detected in any unstressed Camptotheca tissues nor in developing seedlings. These data suggest that tdc1 may be part of a developmentally regulated chemical defense system in Camptotheca , while tdc2 serves as part of a defense system induced during pathogen challenge.     

The rat expresses two complement factor C4 proteins, but only one isotype is expressed in the liver

The complement component C4 is well known for its complex genetics in human and mouse where it is part of a tandemly duplicated module. For the rat, no such information had been available until recently. A C4 gene duplication could be identified also in the rat, but the duplicated module maps approximately 200 kb centromerically from the canonical C4-1 gene. In this study, we present the genomic organization of the two C4 gene-containing modules and the expression of the two C4 genes in the rat (Rattus norvegicus). The duplicated module contains an intact C4 gene as well as Cyp21 and Stk19 pseudogenes. Quantitative mRNA expression analyses revealed that both C4 genes are transcribed in various organs and tissues, but displaying ample differences of C4-1 and C4-2 expression. Most notably, C4-2 is not expressed in the liver. At variance to the mouse, the expression of the rat C4 genes does not exhibit any sex dependency. By using two-dimensional gel electrophoresis and mass spectrometry, products of both C4 genes could be identified in rat serum samples. These two rat C4 isotypes are nearly identical, but differ in a functionally important amino acid residue that is known to influence the functional properties of the C4 isotypes in human.     

Evidence that there are only two tRNA(Phe) genes in Escherichia coli.

pheV, one of the genes that code for tRNA(Phe), was deleted from the chromosome of a strain of Escherichia coli K-12. As a consequence of this mutation, expression of pheA, the gene for chorismate mutase P-prephenate dehydratase, the first enzyme in the terminal pathway of phenylalanine biosynthesis, was derepressed. Similar derepression of pheA has been reported in pheR mutants of E. coli K-12 (J. Gowrishankar and J. Pittard, J. Bacteriol. 150:1130-1137, 1982). Attempts to introduce a pheR mutation into the delta pheV strain failed under circumstances suggesting that this combination of mutations is lethal. Southern blot analysis of pheV+ and delta pheV strains indicated that there are only two tRNA(Phe) genes in E. coli. It is recommended that the names pheU and pheV be retained for these genes.     

The seed lectins of black locust (robinia pseudoacacia) are encoded by two genes which differ from the bark lectin genes

Two lectins were isolated from Robinia pseudoacacia (black locust) seeds using affinity chromatography on fetuin-agarose, and ion exchange chromatography on a Neobar CS column. The first lectin, R. pseudoacacia seed agglutinin I, referred to as RPsAI, is a homotetramer of four 34 kDa subunits whereas the second lectin, referred to as RPsAII, is composed of four 29 kDa polypeptides. cDNA clones encoding the polypeptides of RPsAI and RPsAII were isolated and their sequences were determined. Both polypeptides are translated from mRNAs of ca. 1.2 kb encoding a precursor carrying a signal peptide. Alignment of the deduced amino acid sequences of the different clones indicates that the 34 and 29 kDa seed lectin polypeptides show 95% sequence identity. In spite of this striking homology, the 29 kDa polypeptide has only one putative glycosylation site whereas the 34 kDa subunit has four of these sites. Carbohydrate analysis revealed that the 34 kDa possesses three carbohydrate chains whereas the 29 kDa polypeptide is only partially glycosylated at one site. A comparison of the deduced amino acid sequences of the two seed and three bark lectin polypeptides demonstrated unambiguously that they are encoded by different genes. This implies that five different genes are involved in the control of the expression of the lectins in black locust.Abbreviations LECRPAs cDNA clone encoding Robinia pseudoacacia seed lectin - LoLI Lathyrus ochrus isolectin I - PsA Pisum sativum agglutinin - RPbAI Robinia pseudoacacia bark agglutinin I - RPbAII Robinia pseudoacacia bark agglutinin II - RPsAI Robinia pseudoacacia seed agglutinin I - RPsAII Robinia pseudoacacia seed agglutinin II     

Degradation of ureidoglycolate in French bean (Phaseolus vulgaris) is catalysed by a ubiquitous ureidoglycolate urea-lyase

A ureidoglycolate-degrading activity was analysed in different tissues of French bean (Phaseolus vulgaris L.) plants during development. Activity was detected in all the tissues analysed, although values were very low in seeds before germination and in cotyledons. After radicle emergence, the activity increased due to high activity present in the axes. The highest levels of specific activity were found in developing fruits, from which the enzyme was purified and characterised. This is the first ureidoglycolate-degrading activity that has been purified to homogeneity from a ureide legume. The enzyme was purified 280 fold, and the specific activity for the pure enzyme was 4.4 units mg⁻¹, which corresponds to a turnover number of 1,055 min⁻¹. The native enzyme has a molecular mass of 240 kDa and consists of six identical or similar-sized subunits each of 38 kDa. The activity of the purified enzyme was completely dependent on manganese and asparagine. The enzyme exhibited hyperbolic, Michaelian kinetics for ureidoglycolate with a K _m value of 3.9 mM. This enzyme has been characterised as a ureidoglycolate urea-lyase (EC 4.3.2.3).     

The mop1 (mediator of paramutation1) mutant progressively reactivates one of the two genes encoded by the MuDR transposon in maize

Transposons make up a sizable portion of most genomes, and most organisms have evolved mechanisms to silence them. In maize, silencing of the Mutator family of transposons is associated with methylation of the terminal inverted repeats (TIRs) surrounding the autonomous element and loss of mudrA expression (the transposase) as well as mudrB (a gene involved in insertional activity). We have previously reported that a mutation that suppresses paramutation in maize, mop1, also hypomethylates Mu1 elements and restores somatic activity to silenced MuDR elements. Here, we describe the progressive reactivation of silenced mudrA after several generations in a mop1 background. In mop1 mutants, the TIRA becomes hypomethylated immediately, but mudrA expression and significant somatic reactivation is not observed until silenced MuDR has been exposed to mop1 for several generations. In subsequent generations, individuals that are heterozygous or wild type for the Mop1 allele continue to exhibit hypomethylation at Mu1 and mudrA TIRs as well as somatic activity and high levels of mudrA expression. Thus, mudrA silencing can be progressively and heritably reversed. Conversely, mudrB expression is never restored, its TIR remains methylated, and new insertions of Mu elements are not observed. These data suggest that mudrA and mudrB silencing may be maintained via distinct mechanisms.     

Intracellular distribution and binding state of aluminum in root apices of two common bean (Phaseolus vulgaris) genotypes in relation to Al toxicity

The role of the intracellular distribution and binding state of aluminum (Al) in Al toxicity, using Al exchange and Al fractionation methodologies, were studied in two common bean ( Phaseolus vulgaris L.) genotypes differing in Al resistance. These two genotypes are characterized by a similar initial period (4 h) of Al sensitivity followed by a contrasting recovery period (8–24 h). A higher initial Al accumulation in Quimbaya (Al resistant) in the 5-mm root apex compared with VAX-1 (Al sensitive) could be related to its higher content of unmethylated pectin and thus higher negative charge of the cell walls (CWs). The binding state and cellular distribution of Al in the root apices revealed that the root elongation rate was significantly negatively correlated with the free apoplastic and the stable-bound, not citrate-exchangeable CW Al representing the most important Al fraction in the root apex (80%), but not with the symplastic and the labile-bound, citrate-exchangeable CW Al. It is postulated that the induced and sustained recovery from the initial Al stress in the Al-resistant genotype Quimbaya requires reducing the stable-bound Al in the apoplast thus allowing cell elongation and division to resume.     

A V region determinant (idiotope) expressed at high frequency in B lymphocytes is encoded by a large set of antibody structural genes.

Idiotope Ac38, a V region determinant of the lambda 1 chain-bearing, germ line encoded antibody B1-8, is expressed at high frequency (approximately 1/40) in lambda 1 chain-bearing B cells. Here, we describe the isolation of lambda-positive hybridomas from C57BL/6 mice which had been immunized with antibody Ac38, the antibody recognizing idiotope Ac38. In Northern blot analysis, mRNA isolated from 10 such hybridomas hybridizes with a cDNA probe from the VH gene expressed in the cell line B1-8. Amino acid sequence analysis of the VH regions of four of the hybridoma proteins reveals that they are all derived from related, though distinct, germ line VH genes. In one case the sequence data suggest that extensive somatic mutation has taken place. Only one of the four sequences derives from the same VH gene that is expressed in the cell line B1-8. Together with earlier evidence, the present data demonstrate that the Ac38 idiotope is a marker for at least five VH and three D region genes in the C57BL/6 germ line. This explains the high frequency at which this idiotope is expressed in the B cell population. In addition, our sequence determinations identify two VH genes in the C57BL/6 strain which are closely related (and possibly allelic) to two known BALB/c VH genes. One of these genes is the gene expressed in the BALB/c myeloma MOPC 104E.     

The two myostatin genes of Atlantic salmon (Salmo salar) are expressed in a variety of tissues.

Two myostatin isoforms were identified in Atlantic salmon (Salmo salar) by RT-PCR, and genomic sequences encoding this negative muscle growth factor were for the first time isolated from a nonmammalian species. Salmon myostatin isoform I is transcribed in white skeletal muscle as a 2346-nucleotide mRNA species that encodes a precursor protein of 373 amino acids. Salmon myostatin I shows 93% sequence identity with isoform II which was isolated from white muscle as a partial cDNA sequence of 1409 nucleotides. In contrast to the restricted gene expression of myostatin in mammals, salmon myostatin I and II mRNAs were identified by RT-PCR in multiple tissues, including white muscle, intestine, brain, gills, tongue and eye. In addition, isoform I mRNA was found in red skeletal muscle, heart, spleen, and ovarian tissue. Using polyclonal antibodies against both isoforms, a 55-kDa precursor protein was detected by Western blot analysis in the red and white skeletal muscle, heart, intestine, and brain. Immunoreactive peptides of 35-40 kDa were identified in the gills, tongue, spleen, and head kidney, while the 25-kDa mature myostatin was found in the eye and serum, and in vitro expressed in rabbit reticulocyte lysate. Salmon myostatin was immunohistochemically localized in the sarcoplasma of red and white muscle fibres, in intestinal epithelial cells, at the basis of the branchial primary lamellae, and in odontoblasts and ameloblasts of the tongue teeth. The results indicate that the role of fish myostatin may not be restricted to muscle growth regulation, but may have additional functions similar to the growth/differentiation factor-11 in mammals.     

Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation.

Saccharomyces cerevisiae possesses two glycogenin isoforms (designated as Glg1p and Glg2p) that both contain a conserved tyrosine residue, Tyr232. However, Glg2p possesses an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p, carrying a C-terminal (His6) tag, was produced in Escherichia coli and purified. By tryptic digestion and reversed phase chromatography a peptide (residues 219-246 of the complete Glg2p sequence) was isolated that contained 4-25 glucosyl residues. Following incubation of Glg2p with UDPglucose, more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone, enzymatic hydrolysis of glycosidic bonds and reversed phase chromatography, mono- and diglucosylated peptides having the sequence PNYGYQSSPAM were generated. MS/MS spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide. The formation of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation. Under various experimental conditions Glg2p-producing cells accumulated approximately 30% less glycogen than a control transformed with a Glg2p lacking plasmid. The size distribution of the glycogen and extractable activities of several glycogen-related enzymes were essentially unchanged. As revealed by high performance anion exchange chromatography, the intracellular maltooligosaccharide pattern of the bacterial cells expressing the functional eukaryotic transgene was significantly altered. Thus, the eukaryotic glycogenin appears to be incompatible with the bacterial initiation of glycogen biosynthesis.     

Deletion of follicle-stimulating hormone (FSH) receptor residues encoded by exon one decreases FSH binding and signaling in the rat

The rat FSH receptor (rFSHR) shares considerable homology with the rat LH receptor (rLHR), yet binds human FSH (hFSH) with high fidelity, suggesting that the binding determinant encoded by the rFSHR gene shares no homology with the analogous rLHR primary sequence, thereby affording specificity of ligand binding. Two such regions of primary sequence have been previously identified and studied by peptide challenge tests and immunoneutralization studies. We therefore implemented site-directed mutagenesis to delete the regions S9-N30 and D300-F315 of the mature rFSHR sequence. The mutant receptor (DeltarFSHR) cDNAs were expressed in insect cells. The large deletion DeltarFSHRS9-N30 and a smaller deletion, DeltarFSHRS9-S18, did not bind (125)I-hFSH. However, DeltarFSHRK19-R29 and DeltarFSHRD300-F315 bound (125)I-hFSH with an affinity indistinguishable from wild-type rFSHR. The deletion mutants DeltarFSHR S9-N30 or DeltarFSHRS9-S18 were not detectable on the cell surface by flow cytometry unless cells were sheared. Although (125)I-hFSH binding to DeltarFSHRK19-R29 was normal, this form of the receptor was defective for signal transduction whereas DeltarFSHRD300-F315 was not. Furthermore, neither region seems to be a specificity determinant, since their removal did not result in high-affinity binding of hCG to DeltarFSHR.     

ATP produced by oxidative phosphorylation is channeled toward hexokinase bound to mitochondrial porin (VDAC) in beetroots (Beta vulgaris)

Mitochondrial porins or voltage-dependent anion channels (VDAC) are the main route for solute transport through outer mitochondrial membranes (OMM). In mammals, hexokinase (HK) binds to VDAC, which allows the channeling of ATP synthesized by oxidative phosphorylation toward HK. In plants, although HK has been found associated with OMM, evidence for an interaction with VDAC is scarce. Thus, in this work, we studied the physical and functional interaction between these proteins in beetroot mitochondria. To observe a physical interaction between HK and VDAC, OMM presenting HK activity were prepared from purified mitochondria. Protein complexes were solubilized from OMM with mild detergents and separated by centrifugation in glycerol gradients. Both HK activity and immunodetected VDAC were found in small (9S–13S) and large (>40S) complexes. OMM proteins were also separated according to their hydropathy by serial phase partitioning with Triton X-114. Most of HK activity was found in hydrophobic fractions where VDAC was also present. These results indicated that HK could be bound to VDAC in beetroot mitochondria. The functional interaction of HK with VDAC was demonstrated by observing the effect of apyrase on HK-catalyzed glucose phosphorylation in intact mitochondria. Apyrase, which hydrolyzes freely soluble ATP, competed efficiently with hexokinase for ATP when it was produced outside mitochondria (with PEP and pyruvate kinase), but not when it was produced inside mitochondria by oxidative phosphorylation. These results suggest that HK closely interacts with VDAC in beetroot mitochondria, and that this interaction allows the channeling of respiratory ATP toward HK through VDAC.