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Identification of new signaling components in the Drosophila genome sequence.

The availability of the complete sequence of the Drosophila genome and the assignment of putative reading frames, provides an opportunity to search for new members in families of proteins generating signaling cascades. The six major pathways that dictate patterning were examined: receptor tyrosine kinases, transforming growth factor beta (TGF beta), Wnt, Toll, Hedgehog and Notch. Several new components were identified for the first four pathways, including ligands, receptors, cytoplasmic components and transcription factors. Most notable is the identification of a vascular endothelial growth factor (VEGF) receptor tyrosine kinase, two insulin/insulin growth factor I (IGF I) receptors without cytoplasmic protein kinase domains, and a family of proteins similar to Rhomboid (a protein involved in cleavage of TGF alpha-like ligands). A new TGF beta family ligand, two new Wnts and a Frizzled receptor were also identified. Finally, for the Toll pathway, two new potential Spatzle-like ligands and two new receptors were identified.

Animals↗

Genetic lineage of poorly differentiated gastric carcinoma with a tubular component analysed by comparative genomic hybridization.

Analysis of cell lineage is based on the use of genetic markers inherent to the lineage to be analysed. The breakpoints of unbalanced translocations, and the pattern of chromosomal loss/gain determined by comparative genomic hybridization (CGH), have been previously used to demonstrate lineages in diffuse-type gastric carcinoma. Signet ring cell carcinoma was shown to progress to poorly differentiated adenocarcinoma, and early diffuse-type gastric carcinoma to advanced diffuse-type gastric carcinoma. The present study focuses on poorly differentiated adenocarcinoma with a tubular component to clarify its derivation. CGH and array CGH were applied to DNA extracted from multiple portions of individual tumours and amplified by degenerate oligonucleotide-primed (DOP) PCR and the changes common to the samples in each tumour (stemline changes) were compared between the tumours with and those without a tubular component. Within individual tumours, the samples from the tubular component and those from the other components had common stemline changes and a very similar frequency pattern of chromosomal changes, indicating their common derivation. Frequent stemline changes were 8q+, 7p+, 3q+, 20q+, and 10p+, and these were different from those in the tumours without a tubular component. It was noticed that there were two subgroups in the tumours with a tubular component: one with 5p+, 6p+, 7p+, and 10p+, and the other without these changes. The latter had cytogenetic and clinicopathological features similar to those of the tumours without a tubular component. Analysis of the clonal evolution process by constructing dendrograms for each tumour gave results consistent with the notion that the latter subgroup may derive from signet ring cell carcinoma and the former from tubular adenocarcinoma.

Adenocarcinoma↗

[Advances in studies of genome-specific repetitive DNA sequences in wheat and related species].

In this paper we review recent advances in studies of several aspects of genome specific repetitive DNA sequences in wheat and related species. The available results demonstrate that genome specific repetitive DNA sequences are important components of genome specificity in wheat and related species. Research on genome specific repetitive DNA sequences is essential to the elucidation of genome function. The application of genome specific repetitive DNA sequences will aid molecular cytogenetic studies in wheat and related species and contributes to genetic improvement of common wheat.

English Abstract↗

Molecular characterisation of subgenomic single-stranded and double-stranded DNA forms isolated from plants infected with tomato golden mosaic virus.

A subgenomic single-stranded DNA present in particles of the geminivirus, tomato golden mosaic virus, has been shown by electron microscope heteroduplex mapping and Southern hybridisation analysis to consist of circular molecules, ca. 1.2 kb in size, derived from the smaller of the two genomic DNA components, DNA B, by deletion of open reading frame (ORF) BR1 and the C-terminal portion of ORF BL1. A covalently closed circular, supercoiled, double-stranded form of the subgenomic DNA has been isolated from virus-infected plants and cloned into pEMBL9. Analysis of the sequence of 22 clones across the deletion boundaries revealed only four different deletion boundaries, derived from four different left hand borders and three different right hand borders. Each border was within a region of 11 nucleotides and gave rise to a narrow size range (1248-1261 nucleotides) for the population of 22 subgenomic DNAs. However apparently smaller subgenomic DNAs were sometimes formed when plants were inoculated with cloned subgenomic DNA, or a construct derived from a subgenomic DNA in which a neomycin phosphotransferase gene had been inserted, together with the genomic DNA components. Mechanisms to account for the size, specificity and formation of the subgenomic DNA are discussed.

Base Sequence↗

Molecular characterization of two bipartite geminiviruses causing squash leaf curl disease: role of viral replication and movement functions in determining host range.

The genomes of two distinct, but highly homologous, bipartite geminiviruses have been identified in and cloned from extracts of squash leaf curl diseased field squash. These two squash leaf curl viruses (SqLCVs) have covalently closed, circular single-stranded DNA genomes with the same bipartite component organization characteristic of other whitefly-transmitted geminiviruses. Infectivity studies using virus preparations or cloned viral genomic components on different potential host plants demonstrated that these two SqLCVs have different host range phenotypes which can be explained by specific interactions among the different viral genomic components that act to influence viral replication and systemic movement in the plant. Analysis of Agrobacterium-inoculated leaf discs demonstrated that replication of the restricted virus was rescued in trans by the nonrestricted virus, providing an explanation for the mixtures of viral DNA components often found in particular hosts in the field. Sequence analysis of the common regions of these two SqLCVs identified a 13-base deletion in the restricted virus as compared to the nonrestricted virus, suggesting a potential sequence alteration likely to be involved in their host range phenotypic differences and strengthening the conclusion based on hybridization studies of their close evolutionary relationship. Also identified in the original field squash was a defective viral component which appeared to interfere with movement of the restricted SqLCV in its normally permissive hosts and accounted for another aspect of host range variation observed for this virus.

Base Sequence↗

An emerging phylogenetic core of Archaea: phylogenies of transcription and translation machineries converge following addition of new genome sequences.

BACKGROUND: The concept of a genomic core, defined as the set of genes ubiquitous in all genomes of a monophyletic group, has become crucial in comparative and evolutionary genomics. However, it is still a matter of debate whether lateral gene transfers (LGT) may affect the components of genomic cores, preventing their use to retrace species evolution. We have recently reconstructed the phylogeny of Archaea by using two large concatenated datasets of core proteins involved in translation and transcription, respectively. The resulting trees were largely congruent, showing that informational gene components of the archaeal genomic core belonging to two distinct molecular systems contain a coherent signal for archaeal phylogeny. However, some incongruence remained between the two phylogenies. This may be due either to undetected LGT and/or to a lack of sufficient phylogenetic signal in the datasets. RESULTS: We present evidence strongly favoring of the latter hypothesis. In fact, we have updated our transcription and translation datasets with five new archaeal genomes for a total of 6384 and 2928 amino acid positions, respectively, and 25 taxa. This increase in taxonomic sampling led to the nearly complete convergence of the transcription-based and translation-based trees on a single phylogenetic pattern for archaeal evolution. In fact, only a single incongruence persisted between the two phylogenies. This concerned Methanopyrus kandleri, whose placement remained strongly biased in the transcription tree due to its above average evolutionary rates, and could not be counterbalanced due to the lack of availability of closely related and/or slower-evolving relatives. CONCLUSION: To our knowledge, this is the first report of evidence that the phylogenetic signal harbored by components of the archaeal translation apparatus is confirmed by additional markers belonging to a second molecular system (i.e. transcription). This rules out the risk of circularity when inferring species evolution by small subunit ribosomal RNA and ribosomal protein sequences, since it has been suggested that concerted LGT may affect these markers. Our results strongly support the existence of a core of proteins that has evolved mainly through vertical inheritance in Archaea, and carries a bona fide phylogenetic signal that can be used to retrace the evolutionary history of this domain. The identification and analysis of additional molecular markers not affected by LGT should continue defining the emerging picture of a genuine phylogenetic core for the third domain of life.

Algorithms↗

Nucleotide sequence of one component of the banana bunchy top virus genome contains a putative replicase gene.

One DNA component of the banana bunchy top virus (BBTV) genome was cloned and sequenced. This component is present as a circular, ssDNA in the virions and consists of 1111 nucleotides. It contains one large open reading frame (ORF) of 858 nucleotides in the virion sense; this ORF encodes a putative replicase based on the presence of a dNTP-binding motif (GGEGKT). Two smaller ORFs (249 and 366 nucleotides), in the complementary orientation, could not be assigned any obvious function. Neither of these ORFs had significant sequence homology with any known DNA plant virus gene or gene product. Computer analysis of this component-predicted a strong stem-loop structure in the virion sense putative untranslated region; a nonanucleotide sequence in the loop was nearly identical to the nonanucleotide invariant loop sequence of geminiviruses and coconut foliar decay virus. There is strong evidence that the genome of BBTV consists of more than one component because no ORF was found that would encode a protein the size of the BBTV coat protein. BBTV has some characteristics in common with geminiviruses but cannot be classified as one. Rather, BBTV probably belongs to an undescribed plant virus group which could also include subterranean clover stunt virus and coconut foliar decay virus.

Amino Acid Sequence↗

Repetitive extragenic palindromic sequences: a major component of the bacterial genome.

We describe a remarkably conserved nucleotide sequence, the many copies of which may occupy up to 1% of the genomes of E. coli and S. typhimurium. This sequence, the REP (repetitive extragenic palindromic) sequence, is about 35 nucleotides long, includes an inverted repeat, and can occur singly or in multiple adjacent copies. A possible role for the REP sequences in regulation of gene expression has been thoroughly investigated. While the REP sequences do not appear to modulate differential gene expression within an operon, they can affect the expression of both upstream and downstream genes to a small extent, probably by affecting the rate of mRNA degradation. Possible roles for the REP sequence in mRNA degradation, chromosome structure, and recombination are discussed.

Base Sequence↗

EcoRI-generated reiterated components of the rat genome. I. Sequence of two (92 and 93 bp) related DNA fragments.

The sequence of the 92 and 93 bp long, highly repetitive DNA fragments, isolated from EcoRI digested rat liver DNA, were determined. These fragments, designated 92 and 93, are found in equal abundance, 6.5 x 10(5) copies per haploid genome. J92 and J93 can be distinguished by their differential sensitivity to cleavage by HaeIII and HindIII, respectively, which cut the fragments at 75 and 57 bp from their mutually homologous 5'-ends. J92 and J93 are 38% and 35.4% G + C, respectively, and contain a disproportionate number of triplets complementary to stop codons in all reading frames. Three methylated sites were found in J92 while none could be detected in J93. The sequences around the m5C sites were 5'-Py-Py-m5C-G-Pu-Pu, except for one case where the second Py was replaced by an A. This site appeared to be hemimethylated. When J92 and J93 are placed in register from their mutually homologous 5'-ends, homology is 73% for the first 30 bp region and 63.5% for the total molecule. Thermal melting studies indicate sequence heterogeneity within J92 and J93 from substantial internal base mismatches. The sequences derived are therefore composite averages for the whole molecules. The Cot1/2 for the sequence was measured spectrophotometrically to be 2 x 10(-2) M/s on a DNA phosphorus basis and 2.15 x 10(-4) M/s on a mole fragment basis.

Animals↗

BIS 1, a major component of the cereal genome and a tool for studying genomic organization.

We report the cloning and characterization of an element (BIS 1) in barley. Related sequences were also found in wheat and rye genomes. BIS 1-related sequences may be some of the more frequent of the complex repeats in the barley genome, and their dispersion throughout the barley genome suggests that they are capable of both mobility and amplification. BIS 1 sequences have been used to study the gross structure of the barley genome. These studies indicate that the genome may be formed from larger genomic structures of tens of kilobases which may be repeated. Surprisingly, these studies also show that these large genomic structures also occur in similar proportions and at similar size distribution in the wheat genome.

Blotting, Southern↗

Insulators are fundamental components of the eukaryotic genomes.

The properties of cis-regulatory elements able to influence gene transcription over large distances have led to the hypothesis that elements called insulators should exist to limit the action of enhancers and silencers. During the last decades, insulators have been identified in many eukaryotes from yeast to human. Insulators possess two main properties: (i) they can block enhancer-promoter communication ('enhancer blocker activity'), and (ii) they can prevent the spread of repressive chromatin ('barrier activity'). This review focuses on recent studies designed to elucidate the molecular mechanisms of the insulator function, and gives an overview of the critical role of insulators in nuclear organization and functional identity of chromatin.

Alleles↗

Middle repetitive DNA: a fluid component of the Drosophila genome.

Most of the middle repetitive DNA of Drosophila melanogaster appears to be organized into families of 10-100 repeated elements that are found at scattered locations in the chromosome arms and occupy new chromosomal positions as populations of D. melanogaster diverge. These "nomadic" DNA segments can be identified by an analysis of cDm plasmids, hybrids of ColE1 and segments of randomly sheared D. melanogaster DNA. Eighty cDm plasmids were withdrawn, at random, from a library of approximately 17,000 cDm clones. Fifty-seven of these seem to contain either DNA that is not repeated in the D. melanogaster genome or DNA that has a low repetition frequency. The remaining 23 cDm plasmids contain repetitive sequences. Seventeen of these 23 plasmids contain repetitive sequences that are demonstrably scattered to many chromosomal sites that can be mapped in two D. melanogaster strains, g-1 and g-X11. The repeated elements hybridizing with each of the different Dm segments are at quite different chromosomal locations in these two strains. However, the size of each family of repeated sequences remains fairly constant in both strains. It is proposed that the number of elements in each family has been fixed by selection.

Animals↗

Gene disruptions using P transposable elements: an integral component of the Drosophila genome project.

Biologists require genetic as well as molecular tools to decipher genomic information and ultimately to understand gene function. The Berkeley Drosophila Genome Project is addressing these needs with a massive gene disruption project that uses individual, genetically engineered P transposable elements to target open reading frames throughout the Drosophila genome. DNA flanking the insertions is sequenced, thereby placing an extensive series of genetic markers on the physical genomic map and associating insertions with specific open reading frames and genes. Insertions from the collection now lie within or near most Drosophila genes, greatly reducing the time required to identify new mutations and analyze gene functions. Information revealed from these studies about P element site specificity is being used to target the remaining open reading frames.

Animals↗

Nucleotide sequence of raspberry bushy dwarf virus RNA-2: a bicistronic component of a bipartite genome.

Northern blot analysis with cDNA probes to RNA-3 (1 kb) of raspberry bushy dwarf virus (RBDV) revealed extensive sequence homology with RBDV RNA-2 (2.2 kb). Nucleotide sequencing showed that RNA-2 contains two large open reading frames (ORFs), of 1074 (5' ORF) and 822 (3' ORF) bases. The 3' ORF is virtually identical in sequence to RNA-3, which encodes the Mr 30509 (30K) coat protein. The 5' ORF encodes an Mr 38860 (39K) protein which slightly resembles the 32K protein encoded by RNA-3 of alfalfa mosaic virus (AlMV). RBDV RNA-2 resembles AlMV RNA-3 in being bicistronic and encoding a coat protein at the 3' end. Comparing RNA-1 and RNA-2 of RBDV, only the 18 3'-terminal nucleotides are identical in sequence but the 3'-terminal 71 nucleotides of each RNA species have the potential to form similar stem-loop structures.

Amino Acid Sequence↗

Sequence organization and genomic distribution of the major family of interspersed repeats of mouse DNA.

We have investigated that organization and the distribution of a family of interspersed DNA repeats in the mouse genome. The repeats are at least 5600 base pairs (bp) in size and contain two contiguous BamHI endonuclease fragments, 4000 and 540 bp in size, the larger of which includes a 1350-bp EcoRI fragment studied by previous authors. The repeats are polymorphic in their restriction maps, and represent the major family of interspersed repeats in the mouse genome. The repeats are present almost exclusively in the two light major components of mouse DNA, and the base composition of their large BamHI fragments matches that of those components. The genomic distribution of the repeats is different from that of structural genes, which are present not only in the two light components but also in the two heavy components of mouse DNA. This distribution indicates that the repeats are not involved, at least in any simple way, in the regulation of gene expression.

Animals↗

Characterization of genomic and complementary DNA sequence of human C-reactive protein, and comparison with the complementary DNA sequence of serum amyloid P component.

Complementary and genomic DNA clones corresponding to the human C-reactive protein (CRP) mRNA and structural gene have been analyzed and compared. Nucleotide sequencing of the coding regions of both cDNA and genomic DNA revealed an additional 19 amino acid peptide not described in the published CRP amino acid sequence. The CRP gene contains a single 278 base pair intron within the codon specifying the third residue of mature CRP. The intron contains a repetitive sequence (GT)15G(GT)3 which is similar to structures capable of adopting the Z-DNA form. A comparison of CRP coding and amino acid sequences with those of serum amyloid P component revealed striking overall homology which was not uniform: a region of limited conservation is bounded by two highly conserved regions.

Amino Acid Sequence↗