The Drosophila PROS-28.1 gene is a member of the proteasome gene family

In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-M_r complex possessing proteolytic activity. Screening a Drosophila λgt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene.     

The PROS-35 gene encodes the 35 kd protein subunit of Drosophila melanogaster proteasome.

The proteasome is a multicatalytic proteinase complex composed of nonidentical protein subunits. We have isolated a cDNA clone encoding the 35 kd proteasome subunit of Drosophila melanogaster and propose the designation PROS-35 for the corresponding gene. The deduced amino acid sequence reveals a region of striking homology to a tyrosine phosphorylation site of viral and cellular proteins suggesting a potential regulatory function for the 35 kd subunit within the proteinase complex. Immunocytochemical experiments reveal a tissue-dependent differential distribution of the proteasome between the nucleus and cytoplasm. In addition developmental analysis shows that the proteasome is highly expressed in the CNS of stage-16 embryos and in cardia, ventriculus and ovaries of adult flies. These data suggest a tissue- and development-dependent distribution of the proteasome in D. melanogaster.     

An Arabidopsis gene homologous to mammalian and insect genes encoding the largest proteasome subunit

A gene encoding a protein with extensive homology to the largest subunit of the multicatalytic proteinase complex (proteasome) has been identified in Arabidopsis thaliana. This gene, referred to as AtPSM30, is entirely encompassed within a previously characterized radiation-induced deletion, which may thus provide the first example of a proteasome null mutation in a higher eukaryote. However, the growth rate and fertility of Arabidopsis plants do not appear to be significantly affected by this mutation, even though disruption experiments in yeast have shown that most proteasome subunits are essential. Analysis of mRNA levels in developing seedlings and mature plants indicates that expression of AtPSM30 is differentially regulated during development and is slightly induced in response to stress, as has been observed for proteasome genes in yeast, Drosophila, and mammals. Southern blot analysis indicates that the Arabidopsis genome contains numerous sequences closely related to AtPSM30, consistent with recent reports of at least two other proteasome genes in Arabidopsis. A comparison of the deduced amino acid sequences for all proteasome genes reported to date suggests that multiple proteasome subunits evolved in eukaryotes prior to the divergence of plants and animals.GenBank accession number: M98495     

Characterization and mapping of the gene encoding mouse proteasome subunit DELTA (Lmp19)

The proteasome subunit DELTA is unusually closely related to the major histocompatibility complex (MHC)-linked proteasome subunit, LMP2. The sequence of a mouse cDNA for DELTA confirms that this 22 100 M _r proteasome subunit is highly conserved across species. Sequence analysis of the mouse gene encoding DELTA, designated Lmp19, indicates that it consists of six exons and five introns, similar to the Lmp2 gene. The 5 upstream region lacks a TATA regulatory sequence, which is also absent from proteasome genes isolated from Drosophila. BXD recombinant inbred (RI) mice were used to map the potential chromosomal location of Lmp19, and revealed that the DELTA subunit has related sequences present on two different mouse chromosomes, chromosomes 1 and 11. Typing of 89 progeny from a C57BL/6J X Mus spretus DNA backcross panel (BSS) confirmed the chromosome 1 assignment. Southern hybridization with a polymerase chain reaction-generated Lmp19 intron 2-specific probe indicates that the Lmp19 genomic clone corresponds to the sequence on chromosome 11, and further suggests that the chromosome 1 copy represents a processed pseudogene (Lmp19-ps1).     

An Arabidopsis gene homologous to mammalian and insect genes encoding the largest proteasome subunit

A gene encoding a protein with extensive homology to the largest subunit of the multicatalytic proteinase complex (proteasome) has been identified in Arabidopsis thaliana. This gene, referred to as AtPSM30, is entirely encompassed within a previously characterized radiation-induced deletion, which may thus provide the first example of a proteasome null mutation in a higher eukaryote. However, the growth rate and fertility of Arabidopsis plants do not appear to be significantly affected by this mutation, even though disruption experiments in yeast have shown that most proteasome subunits are essential. Analysis of mRNA levels in developing seedlings and mature plants indicates that expression of AtPSM30 is differentially regulated during development and is slightly induced in response to stress, as has been observed for proteasome genes in yeast, Drosophila, and mammals. Southern blot analysis indicates that the Arabidopsis genome contains numerous sequences closely related to AtPSM30, consistent with recent reports of at least two other proteasome genes in Arabidopsis. A comparison of the deduced amino acid sequences for all proteasome genes reported to date suggests that multiple proteasome subunits evolved in eukaryotes prior to the divergence of plants and animals.     

A Gene Located at 56F1-2 inDrosophila melanogasterEncodes a Novel Metazoan β-like Subunit of Casein Kinase II

Drosophila melanogastercasein kinase II (DmCKII) is composed of catalytic α and regulatory β subunits associated as an α₂β₂heterotetramer. Using the two-hybrid system, we have screened aDrosophilaembryo cDNA library for proteins that interact with DmCKII α. One of the cDNAs encodes a novel β-like polypeptide, which we designate β′.In situhybridization localizes the corresponding gene to 56F1-2, a site distinct from that of both the β gene and theStellatefamily of β-like sequences. The predicted sequence of β′ is more closely related to the β subunit ofDrosophilaand other metazoans than to the Stellate family of proteins, suggesting that it is a second regulatory subunit.In vitroreconstitution studies show that a GST-β′ fusion protein associates with the α subunit to generate a tetrameric complex with regulatory properties similar to those of the native α₂β₂holoenzyme. The data are consistent with the proposed role of the β′ subunit as an integral component of the holoenzyme.     

Ubiquitins (polyubiquitin and ubiquitin extension protein) in marine sponges: cDNA sequence and phylogenetic analysis

The complete nucleotide sequences of two Suberites domuncula cDNAs and one Sycon raphanus cDNA, all encoding ubiquitin, have been determined. One cDNA from S. domuncula codes for polyubiquitin with four tandemly repeated monomeric units and the second cDNA encodes ubiquitin fused to a ribosomal protein of 78 amino acids (aa). S. domuncula possesses at least one additional polyubiquitin gene, from which the last two monomers were also sequenced. All analysed genes from S. domuncula encode identical ubiquitin proteins, with only one aa difference (Ala 19) to the human/higher animals ubiquitin (Pro 19). Ubiquitin in S. domuncula is identical with the ubiquitin found in another Demospongia, Geodia cydonium. The cDNA from S. raphanus encodes polyubiquitin with seven tandemly repeated units. All these gene monomers code for the same ubiquitin, which differs from the human/higher animals ubiquitin only at position 24 (Asp in Sycon, Glu in others). However, ubiquitin from S. raphanus (Calcarea) shows two aa differences (positions 19 and 24), when compared with the ubiquitin sequences from the two Demospongiae. In a phylogenetic tree constructed by multiple sequence alignment of all sponge ubiquitin gene monomers so far identified, all monomers from the same species cluster together, with the clear exception of the monomer from S. domuncula ribosomal protein fusion gene. This monomer branches off first from the tree and forms a separate line; this gives evidence for a very ancient split of ubiquitin-ribosomal-protein fusion genes from polyubiquitin encoding genes and their long separate coexistence in eukaryotes. The ubiquitin extension protein from S. domuncula is 78 aa long, displays all characteristics of 76–81 aa long ribosomal fusion proteins and shows 78% identity in the first 73 aa with the human S27a protein. However, its C-terminal sequence: 69-GLTYVYKKSD-78 is more similar to the plant consensus (69-GLTYVYQ/NK-76), than to the higher animal consensus (69-CLTYCFNK-76). This protein isolated from a sponge, belonging to the phylogenetically oldest multicellular animals, the Porifera, branches off first from the phylogenetic tree of metazoan ubiquitin extension proteins of the small ribosomal subunits.     

Cloning and sequencing of the gene encoding the large (alpha-) subunit of the proteasome from Thermoplasma acidophilum

The gene encoding the alpha-subunit of the proteasome from the archaebacterium Thermoplasma acidophilum was cloned and sequenced. The gene encodes for a polypeptide with 233 amino acid residues and a calculated molecular weight of 25870. Sequence similarity of the alpha-subunit with the Saccharomyces cerevisiae wild-type suppressor gene scll+ encoded polypeptide, which is probably identical with the subunit YC7-alpha of the yeast proteasome, lends support to a putative role of proteasomes in the regulation of gene expression. The significant sequence similarity to the various subunits of eukaryotic proteasomes make it likely that proteasomal proteins are encoded by one gene family of ancient origin.     

Duplicated Proteasome Subunit Genes in Drosophila Melanogaster Encoding Testes-Specific Isoforms

Using the previously cloned proteasome α-type subunit gene Pros28.1, we screened a Drosophila melanogaster genomic library using reduced stringency conditions to identify closely related genes. Two new genes, Pros28.1A (map position 92F) and Pros28.1B (map position 60D7), showing high sequence similarity to Pros28.1, were identified and characterized. Pros28.1A encodes a protein with 74% amino acid identity to PROS28.1, while the Pros28.1B gene product is 58% identical. The Pros28.1B gene has two introns, located in exactly analogous positions as the two introns in Pros28.1, while the Pros28.1A gene lacks introns. Northern blot analysis reveals that the two new genes are expressed only in males, during the pupal and adult stages. Tissue-specific patterns of expression were examined using transgenic flies carrying lacz-fusion reporter genes. This analysis revealed that both genes are expressed in germiline cells during spermatogenesis, although their expression patterns differed. Pros28.1A expression is first detected at the primary spermatocyte stage and persists into the spermatid elongation phase of spermiogenesis, while Pros28.1B expression is prominent only during spermatid elongation. These genes represent the most striking example of cell-type-specific proteasome gene expression reported to date in any system and support the notion that there is structural and functional heterogeneity among proteasomes in metazoans.     

Mg-chelatase of tobacco: identification of a Chl D cDNA sequence encoding a third subunit, analysis of the interaction of the three subunits with the yeast two-hybrid system, and reconstitution of the enzyme activity by co-expression of recombinant CHL D, CHL H and CHL I

Mg-protoporphyrin IX chelatase catalyzes insertion of the magnesium ion into protoporphyrin IX, the last common intermediate precursor in chlorophyll and heme biosynthesis, to form Mg-protoporphyrin IX. In Rhodobacter sphaeroides, and Synechocystis, the three open reading frames bchD/chlD, bchH/chlH and bchl/chll encode proteins which are required for in vitro Mg-chelatase activity. In higher plants also, three proteins are necessary for the Mg chelation, and genes homologous to bchH and bchl have been isolated previously. In this study, a novel tobacco cDNA sequence homologous to bchD is isolated and initially characterized. Together with the tobacco clones encoding the other two subunits, full-length cDNAs are now available for the first time for all three subunits of one plant species. The CHL D polypeptide deduced from the open reading frame encodes a protein of 758 aa (82.9 kDa) with an amino terminal extension that resembles a plastid transit peptide. Sequence comparison of tobacco CHL D revealed similarities to the D subunit of Rhodobacter and Synechocystis of 44% and 75%. The amino terminal half of CHL D shows significant similarity (46%) to the entire CHL I peptide sequence, indicating a gene duplication from an ancestral gene. The carboxy terminal half seemed to be unique. Both parts of CHL D are linked with a glutamine/asparagine/proline-rich region flanked by a highly acid-rich segment. Protein-protein interaction among the three subunits CHL D, H and I was studied using the yeast two-hybrid system. Physical interaction was demonstrated between CHL D and CHL I indicating that CHL D is part of the Mg-chelatase. Heterodimer formation of CHL H with CHL I or CHL D could not be demonstrated by transactivation of the lacZ reporter gene. Homodimerization of the CHL D subunit was indicated in the more sensitive assay on X-Gal-containing agar plates. In vitro Mg²⁺ insertion into protoporphyrin IX was demonstrated in protein extracts of yeast strains expressing the three subunits of tobacco Mg-chelatase. The reconstitution of the recombinant enzyme activity required additional ATP.     

Cloning, sequencing and characterization of the [NiFe]hydrogenase-encoding structural genes (hoxK and hoxG) from Azotobacter vinelandii

The Azotobacter vinelandii [NiFe]hydrogenase-encoding structural genes were isolated from an A. vinelandii genomic cosmid library. Nucleotide (nt) sequence analysis showed that the two genes, hoxK and hoxG, which encode the small and large subunits of the enzyme, respectively, form part of an operon that contains at least one other gene. The hoxK gene encodes a polypeptide of 358 amino acids (aa) (39,209 Da). The deduced aa sequence encodes a possible 45-aa N-terminus extension, not present in the purified A. vinelandii hydrogenase small subunit, which could be a cellular targeting sequence. The hoxG gene is downstream form, and overlaps hoxK by 4 nt and encodes a 602-aa polypeptide of 66,803 Da. The hoxK and hoxG gene products display homology to aa sequences of hydrogenase small and large subunits, respectively, from other organisms. The hoxG gene lies 16 nt upstream from a third open reading frame which could encode a 27,729-Da (240-aa) hydrophobic polypeptide containing 53% nonpolar and 11% aromatic aa. The significance of this possible third gene is not known at present.     

Phylogenomic Analysis of the α Proteasome Gene Family from Early-Diverging Eukaryotes

We employed a phylogenomic approach to study the evolution of α subunits of the proteasome gene family from early diverging eukaryotes. BLAST similarity searches of the Giardia lamblia genome identified all seven α proteasome genes characteristic of eukaryotes from the crown group. In addition, a PCR strategy for the amplification of multiple α subunit sequences generated single α proteasome products for representatives of the Kinetoplastida (Leishmania major), the Parabasalia (Trichomonas vaginalis), and the Microsporidia (Vairimorpha sp., Nosema sp., Endoreticulata sp., and Spraguea lophii). The kinetoplastid Trypanosoma cruzi and the eukaryote crown group Acanthamoeba castellanii yielded two distinct α proteasome genes each. The presence of seven distinct α proteasome genes in G. lamblia, one of the earliest-diverging eukaryotes, indicates that the α proteasome gene family evolved rapidly from a minimum of one gene in Archaea to seven or more in Eukarya. Results from the phylogenomic analysis are consistent with the idea that the Diplomonida (as represented by G. lamblia), the Kinetoplastida, the Parabasalia, and the Microsporidia diverged after the duplication events that originated the α proteasome gene family. A model for the early origin and evolution of the proteasome gene family is presented. Received: 14 February 2000 / Accepted: 14 August 2000     

Structure and expression of the Chlorobium vibrioforme hemA gene

The green sulfur bacterium, Chlorobium vibrioforme, synthesizes the tetrapyrrole precursor, -aminolevulinic acid (ALA), from glutamate via the RNA-dependent five-carbon pathway. A 1.9-kb clone of genomic DNA from C. vibrioforme that is capable of transforming a glutamyl-tRNA reductase-deficient, ALA-dependent, hemA mutant of Escherichia coli to prototrophy was sequenced. The transforming C. vibrioforme DNA has significant sequence similarity to the E. coli, Salmonella typhimurium, and Bacillus subtilis hemA genes and contains a 1245 base open reading frame that encodes a 415 amino acid polypeptide with a calculated molecular weight of 46174. This polypeptide has over 28% amino acid identity with the polypeptides deduced from the nucleic acid sequences of the E. coli, S. typhimurium, and B. subtilis hemA genes. No sequence similarity was detected, at either the nucleic acid or the peptide level, with the Rhodobacter capsulatus or Bradyrhizobium japonicum hemA genes, which encode ALA synthase, or with the S. typhimurium hemL gene, which encodes glutamate-1-semialdehyde aminotransferase. These results establish that hemA encodes glutamyl-tRNA reductase in species that use the five-carbon ALA biosynthetic pathway. A second region of the cloned DNA, located downstream from the hemA gene, has significant sequence similarity to the E. coli and B. subtilis hemC genes. This region contains a potential open reading frame that encodes a polypeptide that has high sequence identity to the deduced E. coli and B. subtilis HemC peptides. hemC encodes the tetrapyrrole biosynthetic enzyme, porphobilinogen deaminase, in these species. Preliminary evidence was obtained for the existence of a 3.0-kb polycistronic meassge that includes the hemA sequence, in exponentially growing C. vibrioforme cells. Results of condon usage analysis for the C. vibrioforme hemA gene indicate that green sulfur bacteria are more closely related to purple nonsulfur bacteria than to enteric bacteria. Sequences corresponding to a polyadenylation signal and a poly(A) attachment site were found immediately downstream from the 3 end of the hemA open reading frame.     

OsPAA2, a distinct alpha 1 subunit gene for the 20S proteasome in rice (Oryza sativa L.)

The 20S proteasome is the proteolytic complex that is involved in removing abnormal proteins and other diverse biological functions. The 20S proteasome is constituted of 28 subunits arranged in four rings of seven subunits, and exists as a hollow cylinder. The two outer rings and the two inner rings are composed of seven different alpha and beta type subunits, respectively, giving an alpha 7 beta 7 beta 7 alpha 7 structure. We previously reported the primary structures of the 14 proteasomal subunit subfamilies in rice (Oryza sativa), representing the first set for all the subfamilies from monocot. In this study, a distinct cDNA sequence encoding the alpha1 subunit, OsPAA2, was identified. The amino acid sequence similarity between the two rice alpha1 subunits was as low as 59.6%, contrasting with those between paralogs of Arabidopsis proteasome subunit genes. The expression pattern of the OsPAA2 gene was different from that of another alpha1 gene, OsPAA1. These data suggest that OsPAA2 might play a distinct role from that of OsPAA1 in the 20S proteasome complex.     

The Intriguing Evolution of the “b” and “G” Subunits in F-type and V-type ATPases: Isolation of the vma-10 Gene from Neurospora crassa

We have characterized the vma-10 gene which encodes the G subunit of the vacuolar ATPase in Neurospora crassa. The gene is somewhat unusual in filamentous fungi because it contains five introns, comprising 71% of the region between the translation start and stop codons. The 5 untranslated region of the gene contains several elements that have been identified in other genes that encode subunits of the vacuolar ATPase in N. crassa. A comparison of G subunits from N. crassa, S. cerevisiae, and animal cells showed that the N-terminal half of the polypeptide shows the highest degree of sequence conservation. Most striking is the observation that this region could form an alpha helix in which all of the conserved residues are clustered on one face. Subunit G appears to be homologous to the b subunit found in F-type ATPases. The major difference between the b and G subunits is the lack of a membrane-spanning region in the G subunit. We have also identified homologous subunits in the operons which encode V-type ATPases in a eubacterium, Enterrococcus hirae, and an archaebacterium, Methanococcus jannaschii. As in eukaryotic vacuolar ATPases the G subunit homologs lack a membrane-spanning region. Although the b and G subunits appear to be derived from a common ancestor, significant changes have evolved. In F-type and V-type ATPases these subunits can have zero, one, or two membrane-spanning regions and can also differ significantly in the number of copies per enzyme.     

Identification and characterization of the gyrB gene from Treponema pallidum subsp. pallidum

The nucleotide sequence of a DNA gyrase B subunit gene (gyrB) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The organization of genes flanking the gyrB gene is unique in comparison to that of other bacteria. The gyrB gene encodes a 637 amino acid protein whose deduced sequence has a high degree of homology with type-II topoisomerase ATPase subunits (GyrB and ParE). Five type-II topoisomerase motifs, an ATP-binding site (Walker A), and amino acid residues that putatively interact with ATP, are highly conserved in the T. pallidum GyrB protein.     

The amino acid sequence of the alpha subunit of mouse salivary androgen-binding protein (ABP), with a comparison to the partial sequence of the beta subunit and to other ligand-binding proteins

It has been suggested that three distinct genes,Abpa, Abpb, andAbpg, determine the three subunits of mouse salivary androgen-binding protein (ABP) (Dlouhy, S. R.,et al., Genetics 115, 535, 1987). We report the putative amino acid sequence of the subunit common to all forms of ABP, the Alpha subunit, and the partial amino acid sequence of the Beta subunit. These sequences have little in common, supporting the notion of at least two distinct genes coding for the subunits of the most common form of salivary ABP, the A:B dimer. A search of GenBank showed that these sequences have not been reported previously. The Beta subunit shows significant homology with helospectin, a member of the glucagon superfamily, but not enough homology to assign it to the family. No homology exists between ABP subunits and members of the ligand-binding carrier family of proteins nor does ABP show homology with other androgen-binding proteins. Particularly interesting is the observation that there is no relationship to rat prostatic steroid binding protein (PBP), given the similarities in protein tertiary structure, the numbers of subunits and their genes, and the earlier observation of ABP cross-reactive material in mouse prostate.Partial support for this work was provided by a PHS AREA award and by the Butler Academic Grants program. Both sources of support are greatly appreciated.A portion of this work constituted partial fulfillment of the honors thesis requirement for Butler University.