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.     

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.     

Endopolygalacturonase is encoded by a multigene family in the basidiomycete Chondrostereum purpureum

The basidiomycete Chondrostereum purpureum produces several plant cell wall-degrading enzymes, including endopolygalacturonase (endoPG). Degenerate oligonucleotide primers were designed according to conserved regions of endoPG genes from various fungi, plants, and bacteria and used to amplify members of this gene family from C. purpureum. Four different amplification products showed significant similarity to known endoPGs and were used as hybridization probes to screen a library of genomic DNA sequences and to retrieve five full-length endoPG genes (epgA, epgB1, epgB2, epgC, and epgD). The identities between the deduced polypeptides for epgA, epgB1, epgC, and epgD ranged from 61.8 to 80.0%, while the deduced polypeptides for epgB1 and epgB2 shared 97.1% identity. Phylogenetic analysis suggested that the duplication of existing endoPG genes occurred after the divergence of the ascomycetes and basidiomycetes. C. purpureum is the first basidiomycete fungus for which the endoPG gene family has been described.     

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.     

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.     

RNA polymerase II large subunit is cleaved by caspases during DNA damage-induced apoptosis

UV radiation induces DNA lesions that are repaired by the nucleotide excision repair (NER) pathway. Cells that are NER deficient such as those derived from xeroderma pigmentosum (XP) patients are susceptible to apoptosis after 10J/m(2) UV radiation, a dose largely survivable by repair proficient cells. Herein, we report that RNA polymerase II large subunit (RNAP II-LS) undergoes caspase-mediated cleavage, yielding a 140kDa C-terminal fragment in XP lymphoblasts but not NER proficient lymphoblasts after 10J/m(2) UV irradiation. Cleavage could also be induced by cisplatin or oxaliplatin, but not transplatin, an isomer of cisplatin that does not induce DNA adducts. The cleavage of RNAP II-LS was blocked by a panel of caspase inhibitors but not by proteasomal inhibitors or inhibitors of other proteases. In vitro cleavage with caspase 8 yielded the same 140kDa RNAP II-LS fragment observed in vivo. Using site-directed mutagenesis, the RNAP II-LS cleavage site was localized to an LETD sequence ending at residue 1339, which is near its C-terminal domain.     

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.     

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.     

In trypanosomes the homolog of the largest subunit of RNA polymerase II is encoded by two genes and has a highly unusual C-terminal domain structure

We have isolated the genes encoding the largest subunit of all three classes of RNA polymerase from Trypanosoma brucei. While the pol II largest subunit is encoded by a single gene in all organisms examined to date, trypanosomes contain two copies of the gene. Both genes are expressed in the procyclic and bloodstream stages of the trypanosome life cycle. The two pol II genes differ from one another in their coding sequences by 21 silent substitutions and 4 amino acid substitutions. In the core part of the large subunit, the predicted polypeptides are similar to other eukaryotic RNA polymerases. Both trypanosome pol II polypeptides, like those of other eukaryotes, also have a unique C-terminal extension. However, this domain in the trypanosome polypeptides, unlike those of other eukaryotes, is not a tandemly repeated heptapeptide sequence.     

The RNA subunit of telomerase is encoded by Marek's disease virus

Marek's disease virus (MDV) is a herpesvirus of chickens that induces T lymphomas and tumors within 4 to 5 weeks of infection. Although the ability of MDV to induce tumors was demonstrated many years ago and although a number of viral oncogenic proteins have been identified, the mechanism by which the MDV is implicated in tumorigenesis is still unknown. We report the identification of a virus-encoded RNA telomerase subunit (vTR) within the genome of MDV. This gene is found in the genomic DNA of the oncogenic MDV strains, whereas it is not carried by the nononcogenic MDV strains. The vTR sequence exhibits 88% sequence identity with the chicken gene (cTR). Our functional analysis suggests that this telomerase RNA can reconstitute telomerase activity in a heterologous system (the knockout murine TR(-/-) cell line) by interacting with the telomerase protein component encoded by the host cell. We have also demonstrated that the vTR promoter region is efficient whatever the species of cell line considered and that vTR is expressed in vivo in peripheral blood leukocytes from chickens infected with the oncogenic MDV-RB1B and the vaccine MDV-Rispens strains. The functionality of the vTR gene and the potential implication of vTR in the oncogenesis induced by MDV is discussed.     

A human cDNA encoding the small subunit of RNA polymerase II transcription factor SIII

A full-length cDNA encoding a human homolog of the 15-kDa subunit (p15) of RNA polymerase II elongation factor SIII was isolated and sequenced. Comparison of the open reading frames of the human p15 cDNA and the previously characterized rat p15 cDNA [Garrett et al., Proc. Natl. Acad. Sci. USA 91 (1994) 5237-5241] indicates that they encode identical proteins and are 93% conserved in nucleotide sequence.