The multifunctional Drosophila melanogaster V-ATPase is encoded by a multigene family.

In animals, V-ATPases are believed to play roles in the plasma membrane, as well as endomembrane. To understand these different functions, it is necessary to adopt a genetic approach in a physiologically tractable model organism. For this purpose, Drosophila melanogaster is ideal, because of the powerful genetics associated with the organism and because of the unusually informative epithelial phenotype provided by the Malpighian tubule. Recently, the first animal "knockouts" of a V-ATPase were described in Drosophila. The resulting phenotypes have general utility for our understanding of V-ATPase function and suggest a screen for novel subunits and associated proteins. Genome project resources have accelerated our knowledge of the V-ATPase gene family size and the new Drosophila genes vhaSFD, vha100-1, vha100-2, vha100-3, vha16-2, vha16-3, vha16-4, vhaPPA1, vhaPPA2, vhaM9.7.1, and vhaM9.7.2 are described. The Drosophila V-ATPase model is thus well-suited to both forward and reverse genetic analysis of this complex multifunctional enzyme.     

Plant inositol monophosphatase is a lithium-sensitive enzyme encoded by a multigene family.

myo-Inositol monophosphatase (IMP) is a soluble, Li(+)-sensitive protein that catalyzes the removal of a phosphate from myo-inositol phosphate substrates. IMP is required for de novo inositol synthesis from glucose 6-phosphate and for breakdown of inositol trisphosphate, a second messenger generated by the phosphatidylinositol signaling pathway. We cloned the IMP gene from tomato (LeIMP) and show that the plant enzyme is encoded by a small gene family. Three different LeIMP cDNAs encode distinct but highly conserved IMP enzymes that are catalytically active in vitro. Similar to the single IMP from animals, the activities of all three LeIMPs are inhibited by low concentrations of LiCl. LeIMP mRNA levels are developmentally regulated in seedlings and fruit and in response to light. Immunoblot analysis detected three proteins of distinct molecular masses (30, 29, and 28 kD) in tomato; these correspond to the predicted molecular masses of the LeIMPs encoded by the genes. Immunoreactive proteins in the same size range are also present in several other plants. Immunolocalization studies indicated that many cell types within seedlings accumulate LeIMP proteins. In particular, cells associated with the vasculature express high levels of LeIMP protein; this may indicate a coordinate regulation between phloem transport and synthesis of inositol. The presence of three distinct enzymes in tomato most likely reflects the complexity of inositol utilization in higher plants.     

The molybdenum-pterin binding protein is encoded by a multigene family in Clostridium pasteurianum

There are three variants of the molybdenum-pterin binding protein (Mop) encoded by three distinct genes in Clostridium pasteurianum. Nucleotide sequence analysis shows that the three mop genes have greater than 90% homology at the nucleotide level. Upstream from the coding region of each mop gene are potential promoter consensus sequences. Analysis of Mop purified from cells grown under nitrogen-fixing conditions indicates all three genes are expressed. Sequence analysis of the three mop genes and the gene products predicts that there are 10 amino acid replacements among the family. The amino acid replacements are chemically conservative accounting for the co-purification of the three variants of Mop. Protein chemistry data suggest the possibility that glutamic acid residues in Mop may be modified in vivo.     

Catalase is encoded by a multigene family in Arabidopsis thaliana (L.) Heynh.

The catalase multigene family in Arabidopsis includes three genes encoding individual subunits that associate to form at least six isozymes that are readily resolved by nondenaturing gel electrophoresis. CAT1 and CAT3 map to chromosome 1, and CAT2 maps to chromosome 4. The nucleotide sequences of the three coding regions are 70 to 72% identical. The amino acid sequences of the three catalase subunits are 75 to 84% identical and 87 to 94% similar, considering conservative substitutions. Both the individual isozymes and the individual subunit mRNAs show distinct patterns of spatial (organ-specific) expression. Six isozymes are detected in flowers and leaves and two are seen in roots. Similarly, mRNA abundance of the three genes varies among organs. All three mRNAs are highly expressed in bolts, and CAT2 and CAT3 are highly expressed in leaves.     

A human RNA polymerase II subunit is encoded by a recently generated multigene family

Background  

Some origins in eukaryotic chromosomes fire more frequently than others. In the fission yeast, Schizosaccharomyces pombe, the relative firing frequencies of the three origins clustered 4-8 kbp upstream of the ura4 gene are controlled by a replication enhancer - an element that stimulates nearby origins in a relatively position-and orientation-independent fashion. The important sequence motifs within this enhancer were not previously localized.     

A human RNA polymerase II subunit is encoded by a recently generated multigene family

Background  

The sequences encoding the yeast RNA polymerase II (RPB) subunits are single copy genes.     

Human cytoplasmic actin proteins are encoded by a multigene family

We characterized nine human actin genes that we isolated (Engel et al., Proc. Natl. Acad. Sci. U.S.A. 78:4674-4678, 1981) from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and alpha-, beta-, and gamma-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria we show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken beta-actin cDNA. We conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.     

Cytosolic ascorbate peroxidase from Arabidopsis thaliana L. is encoded by a small multigene family

A second cytosolic ascorbate peroxidase (cAPX; EC 1.11.1.11) gene from Arabidopsis thaliana has been characterised. This second gene (designated APX1b) maps to linkage group 3 and potentially encodes a cAPX as closely related to that from other dicotyledonous species as to the other member of this gene family (Kubo et al, 1993, FEBS Lett 315: 313–317; here designated APX1a), which maps to linkage group 1. In contrast, the lack of sequence similarity in non-coding regions of the genes implies that they are differentially regulated. Under non-stressed conditions only APX1a is expressed. APX1b was identified during low-stringency probing using a cDNA coding for pea cAPX which, in turn, was recovered from a cDNA library by immunoscreening with an antiserum raised against tea plastidial APX (pAPX). No pAPX cDNAs were recovered, despite the antiserum displaying specificity for pAPX in Western blots.Abbreviations ATG methionine translation initiation codon - bp base pair - cAPX cytosolic ascorbate peroxidase - pAPX plastidial ascorbate peroxidase - RFLP restriction fragment length polymorphism Accession numbers: The APX1b sequence is in the EMBL database under accession number X80036M.S. gratefully acknowledges the support from the Junta Nacional de Investigaçâo Cientifica e Tecnológia, Portugal (grant number BD/394/90-IE). This work was supported by the Biotechnological and Biological Sciences Research Council through a grant-in-aid to the John Innes Centre.     

Halobacterial flagellins are encoded by a multigene family. Characterization of five flagellin genes

Purified flagellar filaments of Halobacterium halobium contain three different protein species based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These proteins were designated as flagellins Fla I, Fla II, and Fla III and were characterized as sulfated glycoproteins with N-glycosidically linked oligosaccharides of the type GlcA-(1----4)-GlcA-(1----4)-GlcA-(1----4)-Glc. All halobacterial flagellin polypeptides are immunologically cross-reactive. A gene fragment of one flagellin was isolated in an expression vector using antibody probes. Using this gene fragment as probe, we identified, subcloned, and determined the nucleotide sequences of five different but highly homologous flagellin genes. Two flagellin (flg) genes are arranged tandemly at one locus (flg A1 and -2), and the other three in a tandem arrangement at a different locus (flg B1, -2, and -3), Two flg mRNAs were detected, one from the A genes and the other from the B genes. Based on immunological analysis, the products of the flg A1 and A2 are Fla II and Fla I, respectively.     

Halobacterial flagellins are encoded by a multigene family. Identification of all five gene products

Flagellins of Halobacterium halobium are encoded in five different but homologous genes. Flagellins isolated from purified flagella were digested and the resulting peptides sequenced. The amino acid sequence data obtained prove that all five gene products are expressed and integrated into the flagellar bundle.     

Breeding Increases the Efficacy of Chondrostereum purpureum in the Sprout Control of Birch

We tested whether the pairing of selected isolates could be used to increase the efficiency of a decay fungus Chondrostereum purpureum (Pers. Ex Fr.) Pouzar to control hardwood sprouting in Finland. We paired C. purpureum strains efficient in sprout control or highly active in laccase production, and tested the efficacy of their progeny in spout control experiments. This procedure resulted in a strain with an efficacy superior to that of the parental strains. The mortality of birch (Betula pendula Roth. and B. pubescens Ehrh.) 1 cm in stump diameter was 78%, 56% and 9% for the best progeny, the best parental strain and the control, respectively. Mortality was only slightly higher for B. pendula than for B. pubescens but no significant differences were found between the number or maximum height of stump sprouts. Our results showed that cross breeding of this decay fungus is a good alternative in attempts to produce efficient biocontrol agents against hardwood sprouting.     

Cytosolic glutamine synthetase in soybean is encoded by a multigene family, and the members are regulated in an organ-specific and developmental manner.

Gln synthetase (GS) is the key enzyme in N metabolism and it catalyzes the synthesis of Gln from glutamic acid, ATP, and NH4+. There are two major isoforms of GS in plants, a cytosolic form (GS1) and a chloroplastic form (GS2). In leaves, GS2 functions to assimilate ammonia produced by nitrate reduction and photorespiration, and GS1 is the major isoform assimilating NH3 produced by all other metabolic processes, including symbiotic N2 fixation in the nodules. GS1 is encoded by a small multigene family in soybean (Glycine max), and cDNA clones for the different members have been isolated. Based on sequence divergence in the 3'-untranslated region, three distinct classes of GS1 genes have been identified (alpha, beta, and gamma). Genomic Southern analysis and analysis of hybrid-select translation products suggest that each class has two distinct members. The alpha forms are the major isoforms in the cotyledons and young roots. The beta forms, although constitutive in their expression pattern, are ammonia inducible and show high expression in N2-fixing nodules. The gamma1 gene appears to be more nodule specific, whereas the gamma2 gene member, although nodule enhanced, is also expressed in the cotyledons and flowers. The two members of the alpha and beta class of GS1 genes show subtle differences in the expression pattern. Analysis of the promoter regions of the gamma1 and gamma2 genes show sequence conservation around the TATA box but complete divergence in the rest of the promoter region. We postulate that each member of the three GS1 gene classes may be derived from the two ancestral genomes from which the allotetraploid soybean was derived.     

The light-harvesting antenna of brown algae: highly homologous proteins encoded by a multigene family.

Accessory light-harvesting complexes (LHCFs) were isolated from the brown alga Laminaria saccharina. Complexes specifically associated with photosystem I or II are identical with each other with respect to molecular mass, isoelectric point and behavior on anion-exchange chromatography or non-denaturing isoelectric focusing. The purified complexes also have similar pigment composition and spectroscopic properties. It is concluded that LHC antennae associated with photosystem I or II cannot be distinguished biochemically. After screening of genomic and cDNA libraries produced from L. saccharina sporophytes, six lhcf genes were isolated. Sequence analysis of these lhcf genes showed a high level of homology between the encoded polypeptides. Comparisons with coding sequences of lhcf genes from Macrocystis pyrifera and expressed sequence tags from Laminaria digitata (two other Laminariales) indicated that these proteins are probably very similar in all brown algae. Another feature common to the lhcf genes characterized was the presence of an intron in the coding region corresponding to the plastid-targeting presequence. The sequence similarity extended to the 5' and 3' UTRs of several genes. In spite of the common origin of the chloroplasts, no light-regulating elements involved in the expression of the genes encoding the higher-plant light-harvesting proteins has been found in the UTRs.     

Genetic variability and structure of Canadian populations of Chondrostereum purpureum,a potential biophytocide

Genetic diversity was studied in four Canadian ecological populations, each corresponding to a Canadian ecozone, of Chondrostereum purpureum, including 93 isolates of various host origin. Pseudo-allelic frequencies were estimated at each of 22 putative RAPD loci by scoring for presence or absence of amplicons in haploid mycelial cultures. The analysis of the hierarchical population structure did not reveal any trend with regard to ecological or host origin. Total gene diversity (H_T? = 0.288) was mostly attributable to diversity within populations (H_S? = 0.269). In addition, the AMOVA analysis detected most of the molecular variability within subpopulations (89.3%; P < 0.001), whereas a significant (7.3%; P = 0.001) proportion of the gene diversity was found among subpopulations, within ecozones. The results indicate that C. purpureum is a highly heterogeneous pathogen with a continuously distributed population across Canada (G_ST? = 0.048), and underscore the importance of considering the population structure in the process of its registration as a microbial control agent. A genotype isolated in either central or eastern Canada (ecozones 2, 4 and 5 populations) and selected for its potential as a biophytocide should be considered indigenous in any of these regions of intended use.     

Immunodetection of protein glycoforms encoded by two independent genes of the self-incompatibility multigene family of brassica

Glycoprotein products of two highly homologous Brassica S gene family members were studied: SLSG (S locus-specific glycoprotein), product of an SLG gene at the S locus, and SLR1 (S locus-related) protein, product of the SLR1 gene, a gene unlinked to the S locus. A polyclonal antibody directed against a trpE-SLR1 fusion protein facilitated study of the SLR1 protein. SLR1 protein was detected in a number of crucifer species. No variation in the level of this protein was found between self-compatible and self-incompatible plants. Both SLSG and SLR1 protein occurred as glycoforms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Each glycoform had several charge forms, indicated by elution patterns from a high performance liquid chromatography cation exchange column and behavior on two-dimensional gels. Deglycosylation of both SLSG and SLR1 protein caused loss of the glycoforms, which apparently arose from differences in glycosylation. Consistent with their apparent similar post-translational processing, immunolocalization showed that SLR1 protein, like SLSG, accumulated in the stigma papillae cell walls. Thus, both SLSG and SLR1 protein are present at the site of pollen-stigma interaction.