The protein sequence homology of gamma-crystallins among major vertebrate classes and their DNA sequence homology to heat-shock protein genes

A systematic characterization of lens crystallins from five major classes of vertebrates was carried out by exclusion gel filtration, cation-exchange chromatography and N-terminal sequence determination. All crystallin fractions except that of -crystallin were found to be N-terminally blocked. -Crystallin is present in major classes of vertebrates except the bird, showing none, or decreased amounts, of this protein in chicken and duck lenses, respectively. N-Terminal sequence analysis of the purified -crystallin polypeptides showed extensive homology between different classes of vertebrates, supporting the close relatedness of this family of crystallin even from the evolutionarily distant species. Comparison of nucleotide sequences and their predicted amino acid sequences between -crystallins of carp and rat lenses and heat-shock proteins demonstrated partial sequence homology of the encoded polypeptides and striking homology at the gene level. The unexpected strong homology of complementary DNA (cDNA) lies in the regions coding for 40 N-terminal residues of carp -II, rat 2-1, and the middle segments of 23,000- and 70,000-M _r heat-shock proteins. The optimal alignment of DNA sequences along these two segments shows about 50% homology. The percentage of protein sequence identity for the corresponding aligned segments is only 20%. The weak sequence homology at the protein level is also found between the invertebrate squid crystallin and rat -crystallin polypeptides. These results pointed to the possibility of unifying three major classes of vertebrate crystallins into one // superfamily and corroborated the previous supposition that the existing crystallins in the animal kingdom are probably mutually interrelated, sharing a common ancestry.     

Characterization of regions of the Caenorhabditis elegans X chromosome containing vitellogenin genes

Caenorhabditis elegans contains a family of vitellogenin genes consisting of five closely related genes (vit-1 to vit-5) coding for 186,000 Mr yolk proteins, and one distantly related gene (vit-6) encoding a 200,000 Mr precursor to two smaller yolk proteins. We demonstrate here that, although vit-1 to vit-5 are not clustered (with the exception of vit-3 and vit-4), they are all on the X chromosome. In contrast, vit-6 is autosomal. The genes are strictly regulated during development: they are activated in the intestine of the hermaphrodite worm, following the last larval molt. In order to determine whether the vit genes are contained within chromosomal domains of similarly regulated genes, we have used the chromosomal "walking" technique to isolate 55,000 to 60,000 base-pairs of DNA surrounding each of the X-linked genes and determined the developmental specificity of nearby genes. In the total of 235,400 base-pairs of cloned DNA, seven genes, in addition to the five vit genes, were found. The average gene spacing is approximately 20,000 base-pairs per gene but is highly variable, ranging from less than 2000 to more than 38,000 base-pairs. The seven newly identified genes, called uvt-1 to uvt-7, specify RNAs varying in size from 500 to 2700 bases. With the exception of uvt-4, all of the genes are developmentally regulated; but the patterns of regulation are quite variable, and all are different from the vitellogenin genes. The vit genes, therefore, are not contained within co-regulated chromosomal domains. We also searched for the presence of repetitive DNA, but only four such sequences were found.     

The role of short sequence repeats in epidemiologic typing.

Short sequence repeats (SSRs), also known as variable number of tandem repeats or micro-satellites, are inherently unstable entities that undergo frequent variation in the number of repeated units through slipped strand mispairing during DNA synthesis. In humans, unit number variability in SSRs has been associated with the occurrence of specific genetic diseases, whereas in micro-organisms SSRs have been elegantly linked to modulation of gene expression. Knowledge of the functional constraints imposed upon the SSRs sheds light on their potential use as molecular clocks for monitoring microbial genome evolution. Although microbial SSR genotypes have been used with increasing frequency for studying the epidemiology and evolution of microbial strains and isolates, such approaches should be used with caution.     

The rDNA of C. elegans: sequence and structure.

We have sequenced one complete rDNA tandem repeat from the nematode C. elegans. By comparative analysis we derive secondary structures for the 18s, 5.8s, and 26s rRNA molecules, and comment on other important features of the sequence. We also present the sequence of a junction between the rDNA and non-ribosomal DNA. Finally, we use our data to quantify the evolutionary relationships among several organisms currently studied in developmental biology.     

The rabbit C family of short, interspersed repeats. Nucleotide sequence determination and transcriptional analysis

When the entire adeno-associated virus (AAV) genome is inserted into a bacterial plasmid, infectious AAV genomes can be rescued and replicated when the recombinant AAV-plasmid DNA is transfected into human 293 cells together with helper adenovirus particles. We have taken advantage of this experimental system to analyze the effects of several classes of mutations on replication of AAV DNA. We obtained AAV mutants by molecular cloning in bacterial plasmids of naturally occurring AAV variant or defective-interfering genomes. Each of these mutants contains a single internal deletion of AAV coding sequences. Also, some of these mutant-AAV plasmids have additional deletions of one or both AAV terminal palindromes introduced during constructions in vitro. We show here that AAV mutants containing internal deletions were defective for replicative form DNA replication (rep-) but could be complemented by intact wild-type AAV. This indicates that an AAV replication function, Rep, is required for normal AAV replication. Mutants in which both terminal palindromes were deleted (ori-) were also replication defective but were not complementable by wild-type AAV. The cis-dominance of the ori- mutation shows that the replication origin is comprised in part of the terminal palindrome. Deletion of only one terminal palindrome was phenotypically wild-type and allowed rescue and replication of AAV genomes in which the deleted region was regenerated apparently by an intramolecular correction mechanism. One model for this correction mechanism is proposed. An AAV ori- mutant also complemented replication of AAV rep- mutants as efficiently as did wild-type AAV. These studies also revealed an unexpected additional property of the deletion mutants in that monomeric single-stranded single-stranded DNA accumulated very inefficiently even though monomeric single-stranded DNA from the complementing wild-type AAV did accumulate.     

lac Up-promoter mutants with increased homology to the consensus promoter sequence.

Four lac promoter mutants were constructed. The mutations increased the homology between the lac promoter and the consensus promoter sequences by introducing the consensus -10 and -35 regions and the consensus spacing of 17 residues between these two regions. The promoter mutants were cloned into a pBR322-derivatized vector upstream from the lacZ gene, and levels of beta-galactosidase were an indication of promoter activity. All mutants exhibited higher activity than did the wild-type promoter.     

Asymmetrically distributed oligonucleotide repeats in the Caenorhabditis elegans genome sequence that map to regions important for meiotic chromosome segregation

The roundworm Caenorhabditis elegans has a haploid karyotype containing six linear chromosomes. The termini of worm chromosomes have been proposed to play an important role in meiotic prophase, either when homologs are participating in a genome-wide search for their proper partners or in the initiation of synapsis. For each chromosome one end appears to stimulate crossing-over with the correct homolog; the other end lacks this property. We have used a bioinformatics approach to identify six repetitive sequence elements in the sequenced C.elegans genome whose distribution closely parallels these putative meiotic pairing centers (MPC) or homolog recognition regions (HRR). We propose that these six DNA sequence elements, which are largely chromosome specific, may correspond to the genetically defined HRR/MPC elements.     

C. elegans: of neurons and genes

The human brain contains 100 billion neurons and probably one thousand times more synapses. Such a system can be analyzed at different complexity levels, from cognitive functions to molecular structure of ion channels. However, it remains extremely difficult to establish links between these different levels. An alternative strategy relies on the use of much simpler animals that can be easily manipulated. In 1974, S. Brenner introduced the nematode Caenorhabditis elegans as a model system. This worm has a simple nervous system that only contains 302 neurons and about 7,000 synapses. Forward genetic screens are powerful tools to identify genes required for specific neuron functions and behaviors. Moreover, studies of mutant phenotypes can identify the function of a protein in the nervous system. The data that have been obtained in C. elegans demonstrate a fascinating conservation of the molecular and cellular biology of the neuron between worms and mammals through more than 550 million years of evolution.     

The long and the short of Wnt signaling in C. elegans

The simplicity of C. elegans makes it an outstanding system to study the role of Wnt signaling in development. Many asymmetric cell divisions in C. elegans require the Wnt/beta-catenin asymmetry pathway. Recent studies confirm that SYS-1 is a structurally and functionally divergent beta-catenin, and implicate lipids and retrograde trafficking in maintenance of WRM-1/beta-catenin asymmetry. Wnts also regulate short-range events such as spindle rotation and gastrulation, and a PCP-like pathway regulates asymmetric divisions. Long-range, cell non-autonomous Wnt signals regulate vulval induction. Both short-range and long-range Wnt signal s are regulated by recycling of MIG-14/Wntless via the retromer complex. These studies indicate that C. elegans continues to be useful for identifying new, conserved mechanisms underlying Wnt signaling in metazoans.     

Conservation of sequence and function of the pag-3 genes from C. elegans and C. briggsae

The Caenorhabditis briggsae homologue of the Caenorhabditis elegans pag-3 gene was cloned and sequenced. When transformed into a C. elegans pag-3 mutant, the C. briggsae pag-3 gene rescued the pag-3 reverse kinker and lethargic phenotypes. The C. elegans pag-3 gene fused to lacZ was expressed in the same pattern in C. elegans and C. briggsae. Unlike many gene homologues compared between C. elegans and C. briggsae, extensive sequence conservation was found in the non-coding regions upstream of the pag-3 exons, in several of the introns and in the downstream non-coding region. Furthermore, the splice acceptor and splice donor sites were conserved, and the size of the introns and exons was surprisingly similar. The predicted protein sequence of C. briggsae PAG-3 was 85% identical to the protein sequence of C. elegans PAG-3. Because so much of the non-coding region of pag-3 was conserved, the control of pag-3 may be quite complex, involving the binding of many trans-acting factors. These results suggest the evolutionary conservation of the pag-3 gene sequence, its expression and function.     

In vivo construction of recombinant molecules within the Caenorhabditis elegans germ line using short regions of terminal homology

Homologous recombination provides a means for the in vivo construction of recombinant DNA molecules that may be problematic to assemble in vitro. We have investigated the efficiency of recombination within the Caenorhabditis elegans germ line as a function of the length of homology between recombining molecules. Our findings indicate that recombination can occur between molecules that share only 10 bp of terminal homology, and that 25 bp is sufficient to mediate relatively high levels of recombination. Recombination occurs with lower efficiency when the location of the homologous segment is subterminal or internal. As in yeast, recombination can also be mediated by either single- or double-stranded bridging oligonucleotides. We find that ligation between cohesive ends is highly efficient and does not require that the ends be phosphorylated; furthermore, precise intermolecular ligation between injected molecules that have blunt ends can also occur within the germ line.     

The contribution of short repeats of low sequence complexity to large conifer genomes

The abundance and genomic organization of six simple sequence repeats, consisting of di-, tri-, and tetranucleotide sequence motifs, and a minisatellite repeat have been analyzed in different gymnosperms by Southern hybridization. Within the gymnosperm genomes investigated, the abundance and genomic organization of micro- and minisatellite repeats largely follows taxonomic groupings. We found that only particular simple sequence repeat motifs are amplified in gymnosperm genomes, while others such as (CAC)₅ and (GACA)₄ are present in only low copy numbers. The variation in abundance of simple sequence motifs reflects a similar situation to that found in angiosperms. Species of the two- and three-needle pine section Pinus are relatively conserved and can be distinguished from Pinus strobus which belongs to the five-needle pine section Strobus. The hybridization pattern of Picea species, bald cypress and gingko were different from the patterns detected in the Pinus species. Furthermore, sequences with homology to the plant telomeric repeat (TTTAGGG)_n have been analyzed in the same set of gymnosperms. Telomere-like repeats are highly amplified within two- and three- needle pine genomes, such as slash pine (Pinus elliottii Engelm. var. elliottii), compared to P. strobus, Picea species, bald cypress and gingko. P. elliottii var. elliottii was used as a representative species to investigate the chromosomal organization of telomere-like sequences by fluorescence in situ hybridization (FISH). The telomere-like sequences are not restricted to the ends of chromosomes; they form large intercalary and pericentric blocks showing that they are a repeated component of the slash pine genome.Conifers have genomes larger than 20000 Mbp, and our results clearly demonstrate that repeats of low sequence complexity, such to (CA)₈, (GA)₈, (GGAT)₄ and (GATA)₄, and minisatellite- and telomere-like sequences represent a large fraction of the repetitive DNA of these species. The striking differences in abundance and genome organization of the various repeat motifs suggest that these repetitive sequences evolved differently in the gymnosperm genomes investigated. Received: 1 October 1999 / Accepted: 3 November 1999     

The DNA sequence at the T7 C promoter.

Restriction fragments of T7 DNA which selectively bind E. coli RNA polymerase have been identified. These include fragments located close to the beginning of gene 1 where according to Minkley and Pribnow (1973) there is a promoter called C. The smallest fragment from this region which binds RNA polymerase has been sequenced. It contains a promoter-like sequence, at an appropriate distance from the sequence TACA which Minkley and Pribnow suggested should lie at the initiation site of C. RNA synthesised in vitro from these fragments has been sequenced. The RNA sequence corresponds to the sequence to the right of the C promoter. The C promoter differs significantly from the A1 A2 and A3 promoters in sequence. Its structure and position suggest it plays a role in T7 infection.     

Functional analyses of vertebrate TCF proteins in C. elegans embryos

In the canonical Wnt pathway, signaling results in the stabilization and increased levels of β-catenin in responding cells. β-catenin then enters the nucleus, functioning as a coactivator for the Wnt effector, TCF/LEF protein. In the absence of Wnt signaling, TCF is complexed with corepressors, together repressing Wnt target genes. In C. elegans, Wnt signaling specifies the E blastomere to become the endoderm precursor. Activation of endoderm genes in E requires not only an increase in β-catenin level, but a concomitant decrease in the nuclear level of POP-1, the sole C. elegans TCF. A decrease in nuclear POP-1 levels requires Wnt-induced phosphorylation of POP-1 and 14-3-3 protein-mediated nuclear export. Nuclear POP-1 levels remain high in the sister cell of E, MS, where POP-1 represses the expression of endoderm genes. Here we express three vertebrate TCF proteins (human TCF4, mouse LEF1 and Xenopus TCF3) in C. elegans embryos and compare their localization, repression and activation functions to POP-1. All three TCFs are localized to the nucleus in C. elegans embryos, but none undergoes Wnt-induced nuclear export. Although unable to undergo Wnt-induced nuclear export, human TCF4, but not mouse LEF1 or Xenopus TCF3, can repress endoderm genes in MS, in a manner very similar to POP-1. This repressive activity requires that human TCF4 recognizes specific promoter sequences upstream of endoderm genes and interacts with C. elegans corepressors. Domain swapping identified two regions of POP-1 that are sufficient to confer nuclear asymmetry to human TCF4 when swapped with its corresponding domains. Despite undergoing Wnt-induced nuclear export, the human TCF4/POP-1 chimeric protein continues to function as a repressor for endoderm genes in E, a result we attribute to the inability of hTCF4 to bind to C. elegans β-catenin. Our results reveal a higher degree of species specificity among TCF proteins for coactivator interactions than for corepressor interactions, and uncover a basic difference between how POP-1 and human TCF4 steady state nuclear levels are regulated.     

Identification and mapping of expressed genes, simple sequence repeats and transposable elements in centromeric regions of rice chromosomes.

The genomic sequences derived from rice centromeric regions were analyzed to facilitate the comprehensive understanding of the rice genome. A rice centromere-specific satellite sequence, RCS2/TrsD/CentO, was used to screen P1-derived artificial chromosome (PAC) and bacterial artificial chromosome (BAC) genomic libraries derived from Oryza sativa L. ssp. japonica cultivar Nipponbare. Physical maps of the centromeric regions were constructed by DNA fingerprinting methods and the aligned clones were analyzed by end sequencing. BLAST analysis revealed the composition of genes, centromeric satellites and other repetitive elements, such as RIRE7/CRR, RIRE8, Squiq, Anaconda, CACTA and miniature inverted-repeat transposable elements. Fiber-fluorescent in situ hybridization analysis also indicated the presence of distinct clusters of RCS2/TrsD/CentO satellite interspersed with other elements, instead of a long homogeneous region. Several expressed genes, sequences representative of ancestral organellar insertions, relatively long simple sequence repeats (SSRs), and sequences corresponding to 5S and 45S ribosomal RNA genes were also identified. Thirty-one gene sequences showed high-similarity to rice full-length cDNA sequences that had not been matched to the published rice genome sequence in silico. These results suggest the presence of expressed genes within and around the clusters of RCS2/TrsD/CentO satellites in unsequenced centromeric regions of the rice chromosomes.