Pea polyubiquitin genes: (I) structure and genomic organization

Four polyubiquitin genes, PUB1, PUB2, PUB3 and PUB4, were isolated from a pea (Pisum sativum L. cv Alaska) genomic library and completely sequenced. They represent all of the four polyubiquitin genes of the ubiquitin gene family in pea. The coding regions of the genes contain five or six coding units arranged as tandem repeats. The different coding repeats of the four genes share homologies between 75 and 97%, encoding the same protein of 76 amino acids identical to those from other higher plants. The open reading frames of PUB1, PUB2 and PUB4 terminate in the additional amino acid, phenylalanine (F), and PUB3 terminates in isoleucine (I). The polyubiquitin genes all contain intron sequences ranging from 584 to 1114 bp immediately 5′ to the ATG initiation codon of the first coding sequence. Of the four genes, three are associated with long AT-rich (85.4–89.4% A+T) sequences ranging from about 331 to 478 bp at their 5′ or 3′ ends. The PUB4 gene was found to be linked to a moderate to highly repetitive DNA at its 5′ flanking sequence. The greater sequence homology between different genes than among individual repeating units of a gene suggests that the polyubiquitin genes may have arisen by gene duplication of a single gene sequence.     

Cloning of a polyubiquitin gene fromNicotiana tabacum and comparison to other polyubiquitin genes

Using a tobacco cDNA clone as a probe, a genomic clone named TUQG-4, coding for a tobacco polyubiquitin protein with the five head-to-tail repeats of ubiquitin monomer was isolated. The five ubiquitin units were completely conserved except for the extra phenylalanine at the carboxy terminus of the last ubiquitin monomer. The putative open reading frame identified from the nucleotide sequence showed two possible intron sequences in the coding region for the first ubiquitin monomer. When the amino acid sequence deduced from the nucleotide sequence of TUQG-4 was compared to the amino acid sequences coded by other polyubiquitin genes of tobacco, there were three or four amino acid differences in the sequence. When the nucleotide sequences coding for the ubiquitin monomers were compared for various species origins, the degree of identity was at the highest between the ubiquitin monomers in one polyubiquitin and did not reflect the distance of the phylogenetic relationship.     

Structure and expression of the polyubiquitin gene in sea urchin embryos.

A cloned Lytechinus pictus cDNA has been identified, which includes seven direct repeats of a 228 bp sequence encoding ubiquitin and about 450 bp of 3' noncoding sequence. The deduced amino acid sequence is identical to that of ubiquitins of other animals (though repeats 3 and 5 each have single amino acid substitutions at different positions). Southern blot analysis revealed that the sea urchin genome contains a single copy of the polyubiquitin gene, and the number of 228 bp repeat units appears to vary from seven to ten among different alleles; no other ubiquitin coding sequences were detected. The size distribution of polyubiquitin mRNA is polymorphic among different individuals, probably corresponding to the differences in copy number of the repetitive coding sequence. The abundance of cytoplasmic polyubiquitin RNA is constant throughout embryogenesis and is similar in ectoderm, endoderm, and mesoderm cells. The constant prevalence of polyubiquitin mRNA apparently results from a balance between ontogenetic changes in its rate of synthesis and its stability in the presence of actinomycin D. Accumulation of polyubiquitin RNA was not heat shock-inducible during embryogenesis.     

Essential factors determining codon usage in ubiquitin genes

Summary Ubiquitin is ubiquitous in all eukaryotes and its amino acid sequence shows extreme conservation. Ubiquitin genes comprise direct repeats of the ubiquitin coding unit with no spacers. The nucleotide sequences coding for 13 ubiquitin genes from 11 species reported so far have been compiled and analyzed. The G+C content of codon third base reveals a positive linear correlation with the genome G+C content of the corresponding species. The slope strongly suggests that the overall G+C content of codons of polyubiquitin genes clearly reflects the genome G+C content by AT/GC substitutions at the codon third position. The G+C content of ubiquitin codon third base also shows a positive linear correlation with the overall G+C content of coding regions of compiled genes, indicating the codon choices among synonymous codons reflect the average codon usage pattern of corresponding species. On the other hand, the monoubiquitin gene, which is different from the polyubiquitin gene in gene organization, gene expression, and function of the encoding protein, shows a different codon usage pattern compared with that of the polyubiquitin gene. From comparisons of the levels of synonymous substitutions among ubiquitin repeats and the homology of the amino acid sequence of the tail of monomeric ubiquitin genes, we propose that the molecular evolution of ubiquitin genes occurred as follows: Plural primitive ubiquitin sequences were dispersed on genome in ancestral eukaryotes. Some of them situated in a particular environment fused with the tail sequence to produce monomeric ubiquitin genes that were maintained across species. After divergence of species, polyubiquitin genes were formed by duplication of the other primitive ubiquitin sequences on different chromosomes. Differences in the environments in which ubiquitin genes are embedded reflect the differences in codon choice and in gene expression pattern between poly- and monomeric ubiquitin genes.     

Characterization of the nucleotide sequence of a polyubiquitin gene (PUBC1) from Arabian camel, Camelus dromedarius

Molecular amplification and sequencing of genomic DNA that encodes camel polyubiquitin (PUBC1) was performed by a polymerase chain reaction (PCR) using various sets of primers. The amplification generated a number of DNA fragments, which were sequenced and compared with the polyubiquitin coding sequences of various species. One DNA fragment that conformed to 325 bp was found to be 95 and 88% homologous to the sequences of human polyubiquitin B and C, respectively. The DNA translated into 108 amino acids that corresponded to two fused units of ubiquitin with no intervening sequence, which indicates that it is a polyubiquitin and contains at least two units of ubiquitin. Although, variations were found in the nucleotide sequence when compared to those of other species, the amino acid sequence was 100% homologous to the polyubiquitin sequences of humans, mice, and rats. This is the first report of the polyubiquitin DNA coding sequence and its corresponding amino acid sequence from camels, amplified using direct genomic DNA preparations.     

Expression Enhancement of a Rice Polyubiquitin Gene Promoter

An 808 bp promoter from a rice polyubiquitin gene, rubi3, has been isolated. The rubi3 gene contained an open reading frame of 1140 bp encoding a pentameric polyubiquitin arranged as five tandem, head-to-tail repeats of 76 aa. The 1140 bp 5′ UTR intron of the gene enhanced its promoter activity in transient expression assays by 20-fold. Translational fusion of the GUS reporter gene to the coding sequence of the ubiquitin monomer enhanced GUS enzyme activity in transient expression assays by 4.3-fold over the construct containing the original rubi3 promoter (including the 5′ UTR intron) construct. The enhancing effect residing in the ubiquitin monomer coding sequence has been narrowed down to the first 9 nt coding for the first three amino acid residues of the ubiquitin protein. Mutagenesis at the third nucleotide of this 9 nt sequence still maintains the enhancing effect, but leads to translation of the native GUS protein rather than a fusion protein. The resultant 5′ regulatory sequence, consisting of the rubi3 promoter, 5′ UTR exon and intron, and the mutated first 9 nt coding sequence, has an activity nearly 90-fold greater than the rubi3 promoter only (without the 5′ UTR intron), and 2.2-fold greater than the maize Ubi1 gene promoter (including its 5′ UTR intron). The newly created expression vector is expected to enhance transgene expression in monocot plants. Considering the high conservation of the polyubiquitin gene structure in higher plants, the observed enhancement in gene expression may apply to 5′ regulatory sequences of other plant polyubiquitin genes.     

Structure and Evolution of Genes Encoding Polyubiquitin and Ubiquitin-like Proteins in Arabidopsis Thaliana Ecotype Columbia

The Arabidopsis thaliana ecotype Columbia ubiquitin gene family consists of 14 members that can be divided into three types of ubiquitin genes; polyubiquitin genes, ubiquitin-like genes and ubiquitin extension genes. The isolation and characterization of eight ubiquitin sequences, consisting of four polyubiquitin genes and four ubiquitin-like genes, are described here, and their relationships to each other and to previously identified Arabidopsis ubiquitin genes were analyzed. The polyubiquitin genes, UBQ3, UBQ10, UBQ11 and UBQ14, contain tandem repeats of the 228-bp ubiquitin coding region. Together with a previously described polyubiquitin gene, UBQ4, they differ in synonymous substitutions, number of ubiquitin coding regions, number and nature of nonubiquitin C-terminal amino acid(s) and chromosomal location, dividing into two subtypes; the UBQ3/UBQ4 and UBQ10/UBQ11/UBQ14 subtypes. Ubiquitin-like genes, UBQ7, UBQ8, UBQ9 and UBQ12, also contain tandem repeats of the ubiquitin coding region, but at least one repeat per gene encodes a protein with amino acid substitutions. Nucleotide comparisons, K(s) value determinations and neighbor-joining analyses were employed to determine intra- and intergenic relationships. In general, the rate of synonymous substitution is too high to discern related repeats. Specific exceptions provide insight into gene relationships. The observed nucleotide relationships are consistent with previously described models involving gene duplications followed by both unequal crossing-over and gene conversion events.     

Molecular cloning and expression of a Tetrahymena pyriformis ubiquitin fusion gene coding for a 53-amino-acid extension protein.

Summary The genome of Tetrahymena pyriformis has been shown to contain a ubiquitin multigene family consisting of several polyubiquitin genes and at least one ubiquitin fusion gene. We report here the isolation and characterization of one genomic clone (pTUl1), that encodes a ubiquitin extension protein. A comparison of the predicted amino acid sequence of the ubiquitin extension protein gene of T. pyriformis with those from other organisms indicated a high degree of homology. However, the Tetrahymena ubiquitin extension protein contains 53 and not 52 amino acids. This feature is different from all ubiquitin 52-amino-acid extension protein genes thus far sequenced. Furthermore, we found an array of four cysteine residues similar to those found in nucleic acid binding proteins. Also, the C-terminal sequence possesses a conserved motif which may represent a nuclear translocation signal. The ubiquitin 53-amino-acid extension protein gene encodes the smallest class of ubiquitin mRNAs in T. pyriformis.     

Evolutionarily conserved structure of the 3' non-translated region of a Chinese hamster polyubiquitin gene.

From a V79 Chinese hamster genomic library, we isolated a clone containing a polyubiquitin gene (designated as CHUB1), and determined its nucleotide sequence. The coding region of the CHUB1 gene consisted of five direct repeats of the ubiquitin unit with no spacer, followed by a single tyrosine residue. Northern hybridization analysis with a synthesized probe specific to the 3' non-translated region of the CHUB1 gene revealed that it codes for a 1.8 kb mRNA. An evident homology to the human polyubiquitin gene UbB and the chicken UbI gene was observed in the region corresponding to the full extent of the mature mRNA sequence, suggesting that these three genes belong to a common polyubiquitin gene subfamily, and that the sequence in the 3' non-translated region of the CHUB1 gene is unique to this subfamily.     

Ubiquitin genes as a paradigm of concerted evolution of tandem repeats

Summary Ubiquitin is remarkable for its ubiquitous distribution and its extreme protein sequence conservation. Ubiquitin genes comprise direct repeats of the ubiquitin coding unit with no spacers. The nucleotide sequences of several ubiquitin repeats from each of humans, chicken,Xenopus, Drosophila, barley, and yeast have recently been determined. By analysis of these data we show that ubiquitin is evolving more slowly than any other known protein, and that this (together with its gene organization) contributes to an ideal situation for the occurrence of concerted evolution of tandem repeats. By contrast, there is little evidence of between-cluster concerted evolution. We deduce that in ubiquitin genes, concerted evolution involves both unequal crossover and gene conversion, and that the average time since two repeated units within the polyubiquitin locus most recently shared a common ancestor is approximately 38 million years (Myr) in mammals, but perhaps only 11 Myr inDrosophila. The extreme conservatism of ubiquitin evolution also allows the inference that certain synonymous serine codons differing at the first two positions were probably mutated at single steps.     

Molecular cloning and characterization of the DNA mismatch repair gene class 2 from the Trypanosoma cruzi

Genes with homology to the bacterial mutS gene, which encodes a protein involved in post-replication DNA mismatch repair, are known in several organisms. Using a degenerate PCR strategy, we cloned a Trypanosoma cruzi genomic DNA fragment homologous to the mutS gene class two (MSH2). This fragment was used as a probe to select the corresponding cDNAs from a T. cruzi cDNA library. The complete sequence of the gene (3304 bp), denominated TcMSH2, was obtained. The sequence contained an open reading frame of 2889 bp coding for a putative protein of 962 amino acids. Computational analyses of the amino acid sequence showed 36% identity with MSH2 proteins from other eukaryotes and revealed the presence of all functional domains of MutS proteins. Hybridization analyses indicated that the TcMSH2 gene is present as a single copy gene that is expressed in all forms of the T. cruzi life cycle. The role of the product of the TcMSH2 gene in mismatch repair was investigated by negative dominance phenotype analyses in Escherichia coli. When eukaryotic muts genes are expressed in a prokaryotic system, they increase the bacterial mutation rate. The same phenomenon was observed with the TcMSH2 cDNA, indicating that T. cruzi MSH2 interferes with the bacterial mismatch system. Phylogenetic analyses showed that the T. cruzi gene grouped with the MSH2 clade confirming the nature of the gene isolated in this work.     

Interspecific Comparison in the Frequency of Concerted Evolution at the Polyubiquitin Gene Locus

The polyubiquitin gene, encoding tandemly repeated multiple ubiquitins, constitutes a uniquitin gene subfamily. It has been demonstrated that polyubiquitin genes are subject to concerted evolution; namely, the individual ubiquitin coding units contained within a polyubiquitin gene are more similar to one another than they are to the ubiquitin coding units in the orthologous gene from other species. However there has been no comprehensive study on the concerted evolution of polyubiquitin genes in a wide range of species, because the relationships (orthologous or paralogous) among multiple polyubiquitin genes from different species have not been extensively analyzed yet. In this report, we present the results of analyzing the nucleotide sequence of polyubiquitin genes of mammals, available in the DDBJ/EMBL/GenBank nucleotide sequence databases, in which we found that there are two groups of polyubiquitin genes in an orthologous relationship. Based on this result, we analyzed the concerted evolution of the polyubiquitin gene in various species and compared the frequency of concerted evolutionary events interspecifically by taking into consideration that the rate of synonymous substitution at the polyubiquitin gene locus may vary depending on species. We found that the concerted evolutionary events in polyubiquitin genes have been more frequent in rats and Chinese hamsters than those in humans, cows, and sheep. The guinea pig polyubiquitin gene was an intermediate example. The frequency of concerted evolution in the mouse gene was unexpectedly low compared to that of other rodent genes. Received: 18 January 2000 / Accepted: 26 April 2000     

Internal structure of the silk fibroin gene of Bombyx mori. II. Remarkable polymorphism of the organization of crystalline and amorphous coding sequences

Alleles of the silk fibroin locus from 22 inbred stocks of Bombyx mori were compared. Nineteen alleles differing from one another in length and internal sequence organization were distinguished. Individuals from a single stock generaly are homozygous for a particular allele, as judged by their gene restriction pattern and the length of the fibroin protein produced. Restriction with endonucleases having four base recognition sequences revealed no variation with respect to these particular coding sequences among the alleles tested. Furthermore, digestion with endonucleases specific for amorphous coding sequences indicated that all the alleles tested had amorphous coding sequence domains alternating regularly with crystalline domains just as was found for the L allele. The stocks differed considerably in their fibroin length, and in the total length of the fibroin coding regions of their genes. These differences were accounted for by variation in the lengths of crystalline coding domains when compared to the ends of the genes. Several characteristics of the alleles indicates that this variation results from recombination between the highly repetitive coding sequences of misaligned genes (homologous unequal crossing-over). Polymorphism of the fibroin gene in B, mori appears to be greater than for any other gene for which data are available.     

Evidence that the entire length of a kinetoplast DNA minicircle is transcribed in Trypanosoma cruzi

A 1.3 kb cDNA (cDNA52) was derived from Trypanosoma cruzi trypomastigote mRNA. Using single stranded probes in Northern blots, we identified the putative coding strand of cDNA52. In addition, a minor band was detected in RNA from epimastigotes that was absent in RNA from trypomastigotes. Nucleotide sequence analysis revealed that cDNA52 was highly homologous to T. cruzi kinetoplast DNA minicircle sequences. All four conserved regions of T. cruzi minicircles were identified in cDNA52. Using several criteria, we demonstrated that the hybridization signals were not caused by contaminating minicircle DNA in the RNA preparations. The data provide direct evidence for the unprecedented finding that the entire length of a kDNA minicircle is transcribed in T. cruzi.     

The human ubiquitin multigene family: some genes contain multiple directly repeated ubiquitin coding sequences.

Ubiquitin coding sequences were isolated from a human genomic library and two cDNA libraries. One human ubiquitin gene consists of 2055 nucleotides and codes for a polyprotein consisting of 685 amino acid residues. The polyprotein contains nine direct repeats of the ubiquitin amino acid sequence and the last ubiquitin sequence is extended with an additional valyl residue at the C-terminal end. No spacer sequences separate the ubiquitin repeats and the coding regions are not interrupted by intervening sequences. This particular gene is transcribed since cDNAs corresponding to the genomic sequence have been isolated. At least two more types of ubiquitin genes are encoded in the human genome, one coding for an ubiquitin monomer while another presumably codes for three or four direct repeats of the ubiquitin sequence. Human DNA contains many copies of the ubiquitin sequence. Ubiquitin is therefore encoded in the human genome as a multigene family.     

Structure and evolution of a mouse tRNA gene cluster encoding tRNAAsp, tRNAGly and tRNAGlu and an unlinked, solitary gene encoding tRNAAsp

We have sequenced mouse tRNA genes from two recombinant lambda phage. An 1800 bp sequence from one phage contains 3 tRNA genes, potentially encoding tRNAAsp, tRNAGly, and tRNAGlu, separated by spacer sequences of 587 bp and 436 bp, respectively. The mouse tRNA gene cluster is homologous to a rat sequence (Sekiya et al., 1981, Nucleic Acids Res. 9, 2239-2250). The mouse and rat tRNAAsp and tRNAGly coding regions are identical. The tRNAGlu coding regions differ at two positions. The flanking sequences contain 3 non-homologous areas: a c. 100 bp insertion in the first mouse spacer, short tandemly repeated sequences in the second spacers and unrelated sequences at the 3' ends of the clusters. In contrast, most of the flanking regions are homologous, consisting of strings of consecutive, identical residues (5-17 bp) separated by single base differences and short insertions/deletions. The latter are often associated with short repeats. The homology of the flanking regions is c. 75%, similar to other murine genes. The second lambda clone contains a solitary mouse tRNAAsp gene. The coding region is identical to that of the clustered tRNAAsp gene. The 5' flanking regions of the two genes contain homologous areas (10-25 bp) separated by unrelated sequences. Overall, the flanking regions of the two mouse tRNAAsp genes are less homologous than those of the mouse and rat clusters.