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PubMed2002

Sequence analysis of the mouse IRBP gene and cDNA.

PURPOSE: To understand the structure of the mouse interphotoreceptor retinoid-binding protein (IRBP) gene and to compare the predicted primary structure within each repeat of IRBP with its relatives. To compare the levels of expression of IRBP RNA in normal and knockout mice. METHODS: The DNA sequence was determined by sub-cloning restriction fragments of the IRBP gene from 129/Sv P1 clones. Primers were designed to utilize the walking approach. Additional sequences were obtained by PCR amplification from genomic DNA and direct sequencing of products. Mouse retina RNA was subjected to reverse transcription coupled to PCR and the accumulation of double stranded DNA product was monitored with SYBR Green. The PCR primers flanked Intron C, to avoid the analysis of contaminating genomic DNA. RESULTS: Altogether a contig was assembled with a final length of about 14.4 kb. The mouse gene structure is similar to the pattern of exons and introns in the bovine and human genes, with a long first exon encoding most of the protein. The splice site boundaries closely match consensus sequences and the exons appear to be identically placed among the three species (bovine, human, and mouse). A region containing a repeated sequence of low complexity is located about 1.75 to 1.4 kb upstream of the transcription start site. A second region containing another low complexity repeat is found in Intron C close to the end of Exon 3. A limited number of weak consensus polyadenylation signals in the 3' region suggest at least three different transcription terminators that apparently give rise to the previously known mouse IRBP mRNAs. The mRNA for IRBP was detected in normal and part of the mRNA was detected in the IRBP knockout mouse, consistent with previous observations. The level of the IRBP mRNA remnant was reduced about 10 fold in the knockout mouse, also consistent with the previously reported absence of Repeat 4 immunoreactivity. CONCLUSIONS: The strong conservation in intron-exon positions, gene structure, and protein sequence among mammals supports an important biological role for these signals and for the IRBP protein in vision. Low levels of aberrant IRBP mRNA in the knockout mouse are consistent with no immunologically detectable Repeat 4 protein in this mouse.

Amino Acid Sequence↗
PubMed2004

Genome sequence of Symbiobacterium thermophilum, an uncultivable bacterium that depends on microbial commensalism.

Symbiobacterium thermophilum is an uncultivable bacterium isolated from compost that depends on microbial commensalism. The 16S ribosomal DNA-based phylogeny suggests that this bacterium belongs to an unknown taxon in the Gram-positive bacterial cluster. Here, we describe the 3.57 Mb genome sequence of S.thermophilum. The genome consists of 3338 protein-coding sequences, out of which 2082 have functional assignments. Despite the high G + C content (68.7%), the genome is closest to that of Firmicutes, a phylum consisting of low G + C Gram-positive bacteria. This provides evidence for the presence of an undefined category in the Gram-positive bacterial group. The presence of both spo and related genes and microscopic observation indicate that S.thermophilum is the first high G + C organism that forms endospores. The S.thermophilum genome is also characterized by the widespread insertion of class C group II introns, which are oriented in the same direction as chromosomal replication. The genome has many membrane transporters, a number of which are involved in the uptake of peptides and amino acids. The genes involved in primary metabolism are largely identified, except those that code several biosynthetic enzymes and carbonic anhydrase. The organism also has a variety of respiratory systems including Nap nitrate reductase, which has been found only in Gram-negative bacteria. Overall, these features suggest that S.thermophilum is adaptable to and thus lives in various environments, such that its growth requirement could be a substance or a physiological condition that is generally available in the natural environment rather than a highly specific substance that is present only in a limited niche. The genomic information from S.thermophilum offers new insights into microbial diversity and evolutionary sciences, and provides a framework for characterizing the molecular basis underlying microbial commensalism.

Actinobacteria↗
PubMed2000

A random sequencing approach for the analysis of the Trypanosoma cruzi genome: general structure, large gene and repetitive DNA families, and gene discovery.

A random sequence survey of the genome of Trypanosoma cruzi, the agent of Chagas disease, was performed and 11,459 genomic sequences were obtained, resulting in approximately 4.3 Mb of readable sequences or approximately 10% of the parasite haploid genome. The estimated total GC content was 50.9%, with a high representation of A and T di- and trinucleotide repeats. Out of the estimated 5000 parasite genes, 947 putative new genes were identified. Another 1723 sequences corresponded to genes detected previously in T. cruzi through expression sequence tag analysis. 7735 sequences had no matches in the database, but the presence of open reading frames that passed Fickett's test suggests that some might contain coding DNA. The survey was highly redundant, with approximately 35% of the sequences included in a few large sequence families. Some of them code for protein families present in dozens of copies, including proteins essential for parasite survival and retrotransposons. Other sequence families include repetitive DNA present in thousands of copies per haploid genome. Some families in the latter group are new, parasite-specific, repetitive DNAs. These results suggest that T. cruzi could constitute an interesting model to analyze gene and genome evolution due to its plasticity in terms of sequence amplification and divergence. Additional information can be found at http://www.iib.unsam.edu.ar/tcruzi.gss. html.

Animals↗
PubMed1978

DNA sequence organization in the soybean plant.

The arrangement of repetitive and nonrepetitive DNA sequences in the soybean genome was ascertained by a comparison of the reassociation kinetics of short (250 nucleotides) and long (2700 nucleotides) DNA fragments, the size distribution of S-1 nuclease resistant repetitive duplexes, and a direct assay of the spectrum of DNA sequences present on long DNA fragments enriched in repetitive DNA. These measurements reveal the following: (1) The 1N genome size of the soybean plant is 1.97 pg. (2) Approximately 40% of the soybean genome consists of nonrepetitive or single-copy DNA sequences, while 60% is repetitive DNA. (3) The repetitive DNA is partitioned into three discrete classes termed "very fast," "fast," and "slow," containing DNA sequences repeated an average of 290,000, 2800, and 19 times each. (4) Approximately 35--50% of the soybean genome is arranged in a short-period interspersion pattern of 250 nucleotide slow sequences and single-copy DNA averaging up to 2700 nucleotides in length. (5) From 30% to 45% of the soybean genome is organized into long stretches of repetitive DNA at least 1500 nucleotides in length. (6) Minimal interspersion of repetitive sequence classes occurs in soybean DNA.

Base Sequence↗
PubMed2002

Pstl repeat: a family of short interspersed nucleotide element (SINE)-like sequences in the genomes of cattle, goat, and buffalo.

The PstI family of elements are short, highly repetitive DNA sequences interspersed throughout the genome of the Bovidae. We have cloned and sequenced some members of the PstI family from cattle, goat, and buffalo. These elements are approximately 500 bp, have a copy number of 2 x 10(5) - 4 x 10(5), and comprise about 4% of the haploid genome. Studies of nucleotide sequence homology indicate that the buffalo and goat PstI repeats (type II) are similar types of short interspersed nucleotide element (SINE) sequences, but the cattle PstI repeat (type I) is considerably more divergent. Additionally, the goat PstI sequence showed significant sequence homology with bovine serine tRNA, and is therefore likely derived from serine tRNA. Interestingly, Southern hybridization suggests that both types of SINEs (I and II) are present in all the species of Bovidae. Dendrogram analysis indicates that cattle PstI SINE is similar to bovine Alu-like SINEs. Goat and buffalo SINEs formed a separate cluster, suggesting that these two types of SINEs evolved separately in the genome of the Bovidae.

Animals↗
PubMed2003

High-Cot sequence analysis of the maize genome.

Higher eukaryotic genomes, including those from plants, contain large amounts of repetitive DNA that complicate genome analysis. We have developed a technique based on DNA renaturation which normalizes repetitive DNA, and thereby allows a more efficient outcome for full genome shotgun sequencing. The data indicate that sequencing the unrenatured outcome of a Cot experiment, otherwise known as High-Cot DNA, enriches genic sequences by more than fourfold in maize, from 5% for a random library to more than 20% for a High-Cot library. Using this approach, we predict that gene discovery would be greater than 95% and that the number of sequencing runs required to sequence the full gene space in maize would be at least fourfold lower than that required for full-genome shotgun sequencing.

Base Sequence↗
PubMed1993

Characterization of human bone marrow-derived closed circular DNA clones.

Because of interest in mechanisms of recombination involved in chromosomal deletions in neoplastic disease, and their relation to possible rearrangements in normal tissues, we are studying circular DNA molecules from human tissue with a long-term goal of investigating them as possible by-products of physiologically relevant intrachromosomal recombination events. Covalently closed circular (ccc) DNA from human bone marrow was cloned in bacteriophage vectors, and fourteen clones chosen randomly from the cccDNA-derived library were characterized. Five clones originated from chromosome-specific centromeric alpha-satellite DNA; two clones carried highly repetitive sequences probably derived from interspersed repetitive elements; six clones were derived from single-copy chromosome-specific sequences which detected homologous rodent sequences; and one clone (EPM10) was derived from a small chromosome 11-specific sequence family which localized to chromosome regions 11cen and 11q14. Oligonucleotide primers derived from the cccDNA clones were used in polymerase chain reaction studies to show that (1) the EPM10 clone carried the circular junction, (2) several of the single-copy products could be detected in three different bone marrow cccDNA preparations, and (3) the Alu-PCR profile for bone marrow cccDNA showed distinct bands which were similar in four bone marrow cccDNA preparations.

Acute Disease↗
PubMed2001

The complex repeats of Dictyostelium discoideum.

In the course of determining the sequence of the Dictyostelium discoideum genome we have characterized in detail the quantity and nature of interspersed repetitive elements present in this species. Several of the most abundant small complex repeats and transposons (DIRS-1; TRE3-A,B; TRE5-A; skipper; Tdd-4; H3R) have been described previously. In our analysis we have identified additional elements. Thus, we can now present a complete list of complex repetitive elements in D. discoideum. All elements add up to 10% of the genome. Some of the newly described elements belong to established classes (TRE3-C, D; TRE5-B,C; DGLT-A,P; Tdd-5). However, we have also defined two new classes of DNA transposable elements (DDT and thug) that have not been described thus far. Based on the nucleotide amount, we calculated the least copy number in each family. These vary between <10 up to >200 copies. Unique sequences adjacent to the element ends and truncation points in elements gave a measure for the fragmentation of the elements. Furthermore, we describe the diversity of single elements with regard to polymorphisms and conserved structures. All elements show insertion preference into loci in which other elements of the same family reside. The analysis of the complex repeats is a valuable data resource for the ongoing assembly of whole D. discoideum chromosomes.

Animals↗
PubMedDate not supplied

L1 family of repetitive DNA sequences in primates may be derived from a sequence encoding a reverse transcriptase-related protein.

Primate and rodent genomes contain a family of highly repetitive, long interspersed sequences, designated the L1 family or LINE-1. Characteristic features of the L1 family sequences such as an A-rich stretch at the 3' end, a truncated 5' end, the existence of significantly long open reading frames (ORFs) and the presence of L1 family transcripts in various types of cells, including pluripotential embryonic cells, suggest that the L1 family is derived from a sequence encoding a protein(s) and dispersed in the genome through an RNA-mediated process. These features of the L1 family are believed to be due to reverse transcription beginning at the 3' end of the L1 transcript and terminating prematurely and to the site duplication caused by the insertion of the complementary DNA. It is likely that this type of transcript is converted to cDNA and inserted into the chromosome through a process similar to that of the formation of processed pseudogenes. The above model, however, does not necessarily explain why the L1 family should produce the extraordinarily large number of copies (more than 10(4) per haploid genome) seen during evolution. It seems likely that the progenitor of the L1 family itself carries (or carried) a function which promotes the active dispersion of the L1 family sequence. We reasoned that such a function, if present, must be conserved during evolution and may be shown by comparative analysis of L1 family sequences from evolutionarily distant species. We show here that the L1 family sequence contains an ORF possessing significant sequence homology to several RNA-dependent DNA polymerases of viral and transposable element origins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2006

How genomes rearrange: genome comparison within bacteria Neisseria suggests roles for mobile elements in formation of complex genome polymorphisms.

Comparison of closely related genome sequences can provide a clue as to how macroscopic genome polymorphisms were formed through various events of recombination. However, this approach has been limited to relatively simple polymorphisms such as insertion, deletion and inversion. In the present study, we tried to extend this approach to more complex genome polymorphisms that were observed when four genome sequences of bacterial genus Neisseria were compared. The first polymorphism was an apparent translocation (ab-cd to cd-ba; a region 'ab' was translocated). The second one was a re-ordering of adjacent regions (ab-cd-ef-gh to ef-cd-ab-gh; ab, cd and ef were in reverse order). The third one was a translocation of two adjacent regions with permutation of their order (ab-cd to cd-ab elsewhere in the genome). The fourth one was a genome-wide inversion associated with a genome-specific insertion into the joints (-ab-cd- to -y-ba-x-cd-). We were able to explain their formation by only a few steps of plausible events of recombination that involved linked IS copies and prophages. Our approach would help to reconstruct a history of apparently complex genome polymorphisms in any forms of organisms and to understand genome rearrangements in the natural environments in non-model organisms.

Base Sequence↗
PubMed2005

Further analysis of VNTR and MIRU in the genome of Mycobacterium avium complex, and application to molecular epidemiology of isolates from South America.

All members of Mycobacterium avium complex are serious pathogens for humans and animals. The aim of this study was to look for and analyze VNTR-MIRU loci in the genome of M. avium complex and their preliminary application to test these isolates. In the present study, we identified 22 novel VNTR-MIRU by using Tandem Repeat software: five with a structure similar to MIRU and 17 without MIRU structure; these latter were designated as VNTR. Most VNTR were located within predicted coding regions. Most MIRU were intercistronic with their extremities overlapping the termination and initiation codons of their flanking genes. Some of these VNTR-MIRU exhibited polymorphism among M. avium complex isolates due to insertion or deletion of whole repeats and/or of nucleotide sequence degeneration. We determined the variability of six VNTR-MIRU loci in 21 M. avium subsp. hominissuis and 26 M. avium subsp. paratuberculosis. The analysis identified 15 different alleles with the combination of six VNTR-MIRU in the 21 M. avium subsp. hominissuis with 16 different IS1245 RFLP and four different profiles with PCR-restriction analysis of hsp65 (PRA). However, neither the six VNTR-MIRU loci nor the PRA were able to distinguish M. avium subsp. paratuberculosis isolates with five different IS900 RFLP profiles. In conclusion, some of the VNTR-MIRU loci identified were useful to differentiate M. avium subsp. hominissuis but not M. avium subsp. paratuberculosis isolates here included. However, we observed polymorphism in VNTR-MIRU loci between M. avium subsp. hominissuis and M. avium subsp. paratuberculosis genomes, which could be important in the understanding of the obvious differences in the pathogenic effects of these mycobacteria.

Animals↗
PubMed1990

On the possibility of obtaining a physical map of genomes by photoelectron imaging.

Photoelectron imaging provides the possibility of a new method of mapping chromosomes. The basic concept is to cause DNA to emit electrons under the action of UV light. The criteria which must be met to map genomes by photoelectron imaging are set forth and discussed. Forming an image of the DNA by accelerating and focusing the electrons is a necessary but not sufficient condition for genome mapping. Equally important is to identify wavelengths of UV light which will cause selective emission from the base pairs, adenine-thymine and guanine-cytosine. The resulting image would then contain a modulation in the image brightness along the DNA duplex. By examining the photoelectron current from uniform films of homopolymers, a wavelength region is identified where marked differences in emission from base pairs is observed. At 160 nm, for example, the relative electron emission from a film of poly(dGdC) is approximately 5 times greater than for an equivalent film of poly(dAdT). Using the experimental data and known sequences, photoelectron gene maps are calculated for the bacteriophage lambda and for a short interspersed repetitive DNA sequence (an Alu repeat) of the human genome. The results suggest that a 5-nm physical map of chromosomes generated by photoelectron imaging would be informative and useful in mapping human and other large genomes.

Bacteriophage lambda↗
PubMedDate not supplied

Genomic data analysis using DNA structure: an analysis of conserved nongenic sequences and ultraconserved elements.

Recent comparative studies of the human and mouse genomes have revealed sets of conserved nongenic sequences (CNGs) and sets of ultraconserved elements (UCEs). Both sets of sequences, which exhibit extremely high levels of conservation, extend over hundreds of bases and have no known function. Since there is no detectable sequence homology between paralogous CNGs or UCEs in either of the species, an alignment-free technique is needed for their analysis. We have previously compiled a database of the structural properties of all 32,896 unique DNA octamers, including information on stability, the minimum energy conformation, and flexibility. We have used Fourier techniques to analyze the UCEs and CNGs in terms of their octamer structural properties, to reveal structural correlations which may indicate possible functions for some of these sequences.

Animals↗
PubMed2005

Progressive proximal expansion of the primate X chromosome centromere.

Previous studies of the pericentromeric region of the human X chromosome short arm (Xp) revealed an age gradient from ancient DNA that contains expressed genes to recent human-specific DNA at the functional centromere. We analyzed the finished sequence of this human genomic region to investigate its evolutionary history. Phylogenetic analysis of >1,500 alpha-satellite monomers from the region revealed the presence of five physical domains, each containing monomers from a distinct phylogenetic clade. The most distal domain contains long interspersed nucleotide element repeats that were active >35 million years ago, whereas the four proximal domains contain more recently active long interspersed nucleotide element repeats. An out-of-register, unequal recombination (i.e., crossover) detected at the edge of the X chromosome-specific alpha-satellite array (DXZ1) may reflect the most recent of a series of punctuating events during evolution that resulted in a proximal physical expansion of the X centromere. The first 18 kb of this array has 97-99% pairwise identity among all 2-kb repeat units. To perform more detailed evolutionary comparisons, we sequenced the junction between the ancient DNA of Xp and the primate-specific alpha satellite in chimpanzee, gorilla, orangutan, vervet, macaque, and baboon. The striking conservation found in all cases supports the ancestral nature of the alpha satellite at this location. These studies demonstrate that the primate X centromere appears to have evolved through repeated expansion events occurring within the central, active region of centromeric DNA, with the newly added sequences then conferring centromere function.

Animals↗
PubMed2006

The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution.

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is a representative of the group of lactic acid-producing bacteria, mainly known for its worldwide application in yogurt production. The genome sequence of this bacterium has been determined and shows the signs of ongoing specialization, with a substantial number of pseudogenes and incomplete metabolic pathways and relatively few regulatory functions. Several unique features of the L. bulgaricus genome support the hypothesis that the genome is in a phase of rapid evolution. (i) Exceptionally high numbers of rRNA and tRNA genes with regard to genome size may indicate that the L. bulgaricus genome has known a recent phase of important size reduction, in agreement with the observed high frequency of gene inactivation and elimination; (ii) a much higher GC content at codon position 3 than expected on the basis of the overall GC content suggests that the composition of the genome is evolving toward a higher GC content; and (iii) the presence of a 47.5-kbp inverted repeat in the replication termination region, an extremely rare feature in bacterial genomes, may be interpreted as a transient stage in genome evolution. The results indicate the adaptation of L. bulgaricus from a plant-associated habitat to the stable protein and lactose-rich milk environment through the loss of superfluous functions and protocooperation with Streptococcus thermophilus.

Bacterial Proteins↗
PubMed2006

Impact of the excision of an ancient repeat insertion on Rickettsia conorii guanylate kinase activity.

The genomic sequencing of Rickettsia conorii revealed a new family of Rickettsia-specific palindromic elements (RPEs) capable of in-frame insertion in preexisting open reading frames (ORFs). Many of these altered ORFs correspond to proteins with well-characterized or essential functions in other microorganisms. Previous experiments indicated that RPE-containing genes are normally transcribed and that no excision of the repeat occurs at the mRNA level. Using mass spectrometry, we now confirmed the retention of the RPE-derived amino acid residues in 4 proteins successfully expressed in Escherichia coli, raising the general question of the consequences of this common insertion event on the fitness of Rickettsia enzymes. The predicted guanylate kinase activity of the R. conorii gmk gene product was measured both on the RPE-containing and RPE-excised recombinant proteins. We show that the 2 proteins are active but exhibit substantial differences in their affinity for adenosine triphosphate, guanosine monophosphate, and catalytic constants. The distribution of the RPEgmk insert among Rickettsia species indicates that the insertion event is ancient and occurred after the divergence of Rickettsia felis and R. conorii but before that of Rickettsia helvetica and R. conorii. We found no evidence that the gmk gene fixed adaptive changes to compensate the RPE peptide insertion. Furthermore, the analysis of the rates of divergence in 23 RPE-containing genes indicates that coding RPE repeats tend to evolve under weak selective constraint, at a rate similar to intergenic noncoding RPE sequences. Altogether, these results suggest that the insertion of RPE-encoded "selfish peptides," although respecting the original fold and activity of the host proteins, might be slightly detrimental to the enzyme efficiency within limits tolerable for slow-growing intracellular parasites such as Rickettsia.

Amino Acid Sequence↗