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PubMed1992

Structure and expression of kin2, one of two cold- and ABA-induced genes of Arabidopsis thaliana.

We report the isolation of the second member, kin2, of a family of two cold-inducible genes of Arabidopsis thaliana. The proteins corresponding to the two genes have similarities to the small antifreeze proteins from Winter flounder. Kin1 and kin2 are organized in a close tandem array in the genome of A. thaliana. Both have three exons separated by introns with approximately the same length and location. The coding regions are highly conserved while the introns and especially the 3' flanking sequences of the mRNAs have diverged. The kin1 and kin2 genes are coordinately regulated in the cold. Unlike kin1, the kin2 mRNA has a detectable basal level, and accumulates to a higher level during acclimation. Both mRNAs are induced by 10 microM ABA but only kin2 responds strongly to drought and salinity stresses.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and complete nucleotide sequence of the repeated unit and flanking gene of the scallop Pecten maximus mitochondrial DNA: putative replication origin features.

In the bivalve mollusc Pecten maximus, the size of the mitochondrial DNA molecules ranges from 20 to 25.8 kbp. This variability is mainly correlated with the occurrence of a variable domain composed with two to five 1.6-kbp repeated units tandemly arrayed in the genome. DNA fragments spanning the 1,586-base-pair-long repeated element and the nearest flanking gene have been cloned and sequenced. This sequence was analyzed regarding its base composition and potential secondary structures. The repeated unit domain was positioned and oriented with regard to the known flanking gene. It ends 2 base pairs upstream relative to the beginning of the tRNAgly gene. The peculiar properties of the repeated unit were compared with those of the 1,442-bp repeated element found in the mitochondrial genome of the deep sea scallop Placopecten magellanicus. This comparison provided evidence for the absence of nucleotide conservation, except for a small sequence engaged in a secondary structure, but argued for a strong pressure maintaining domains with specific nucleotide content. A possible role for the conserved sequence is discussed.

Animals↗

PubMed1996

Stage-specific expression of the Leishmania mexicana paraflagellar rod protein PFR-2.

A screen for Leishmania mexicana genes encoding promastigote-specific flagellar proteins resulted in isolation of genes encoding the major components of the paraflagellar rod. One of these, PFR-2, was characterized extensively. PFR-2 genes are present in the genome as a tandem array of three genes designated PFR-2A, PFR-2B, PFR-2C. PFR-2A and PFR-2B are encoded by a 3.1 transcript while PFR-2C is encoded by a 3.8-kb transcript that has a 3' UTR different from that of the 3.1-kb transcript. Both of these mRNAs were 15-fold more abundant in promastigotes than in amastigotes. Two transcripts immediately upstream of the locus were constitutively expressed while two downstream transcripts were fourfold more abundant in promastigotes than in amastigotes. The PFR genes will provide a good model system for analysis of stage-specific gene regulation in Leishmania as well as assist in the characterization of the function and organization of the paraflagellar rod.

Amino Acid Sequence↗

PubMed1994

Characterization of a satellite DNA from Antilocapra americana.

The nucleotide sequence of a cloned satellite DNA from Antilocapra americana (American pronghorn antelope) is presented. The 1477-bp satellite is composed of degenerate 31-bp sub-repeats which are very similar in sequence to those of the major satellite DNAs from cattle and sheep. The sub-repeat sequence is more degenerate and variable in pronghorn than it is in cattle or sheep. The sequence is organized in the pronghorn genome in multicopy tandem arrays.

Animals↗

PubMed2005

Metacaspases of Trypanosoma cruzi: possible candidates for programmed cell death mediators.

The genome of Trypanosoma cruzi, the Protozoan parasite causing the American Trypanosomiasis, Chagas disease, contains two genes, TcMCA3 and TcMCA5, with homology to those encoding metacaspases, distantly related to the caspases involved in programmed cell death (PCD) in higher eukaryotes. TcMCA3 is present in the CL Brener clone at 16 copies per haploid genome, arrayed in two tandems located in chromosomes of 0.54 and 0.98 Mbp. TcMCA5, on the other hand, is present as a single copy gene. The proteins encoded were expressed in Escherichia coli BL21 [DE3] cells, and used to generate antibodies, which allowed demonstrating that TcMCA3 is expressed in the four major developmental stages of the parasite, whereas TcMCA5 is expressed only in the epimastigote form. Moreover, recombinant TcMCA3, but not TcMCA5, was recognized by most sera from chronic Chagasic patients, showing that the protein is expressed during natural infections. All attempts to show processing and enzyme activity in the recombinant proteins have been unsuccessful so far; however, indirect evidence suggests that the metacaspases might be involved in PCD of the parasite. (1) Immunofluorescence experiments showed that both proteins change their subcellular localization during fresh human serum (FHS)-induced PCD migrating into the nucleus. (2) Epimastigotes over-expressing TcMCA5 were more sensitive to FHS-induced PCD than the controls. (3) PCD was parallelled by an increase in peptidase activity against Z-YVAD-AFC, a typical caspase substrate, and the apoptotic nuclei cells were labeled in vivo with the pan-caspase fluorescent inhibitor SR-VAD-FMK. Further experiments will be required to complete the characterization of these proteins and elucidate their role in the parasite.

Animals↗

PubMed1998

Cloning, expression and reconstitution of the trypanothione-dependent peroxidase system of Crithidia fasciculata.

As a consequence of aerobic metabolism, trypanosomatids are exposed to reactive oxygen intermediates such as superoxide, hydrogen peroxide and the hydroxyl radical. Metabolism of hydrogen peroxide in Crithidia fasciculata is accomplished by three distinct proteins, tryparedoxin, tryparedoxin peroxidase and trypanothione reductase, working in concert with the substrates NADPH and trypanothione. Here, we report the cloning and characterisation of the tryparedoxin (TryX) and tryparedoxin peroxidase (TryP) genes from C. fasciculata. Both genes are multicopy and organized in distinct tandem arrays in the genome. TryX encodes a 16 kDa protein, which belongs to the thioredoxin superfamily, sharing the WCPPC motif, whereas TryP encodes a 21 kDa protein belonging to a new class of peroxidases called 2-Cys peroxidoxins. Both TryX and TryP were expressed in Escherichia coli and the purified recombinant proteins shown to utilise hydrogen peroxide in the presence of NADPH, trypanothione and trypanothione reductase, similar to the native proteins. TryX is rapidly reduced by trypanothione, but weakly by glutathionylspermidine, glutathione or ovothiol A. TryP shows a broad substrate specificity and can reduced hydrogen peroxide, t-butyl hydroperoxide and cumene hydroperoxide with equal efficiency.

Amino Acid Sequence↗

PubMed1980

Satellite DNA is transcribed on lampbrush chromosomes.

During the lampbrush stage of oogenesis there is widespread transcription, and it has been estimated that the total amount of DNA transcribed may be an order of magnitude greater than that required to produce the necessary functional RNA for the oocyte. We therefore considered it likely that some of the transcribed sequences have little, if any, translational significance, and may include both middle repetitive and highly repeated, or satellite, sequences. Satellite DNA is generally defined as rapidly reannealing DNA which has a short basic sequence that is repeated millions of times in the genome, usually in tandem arrays. The short repeated length, coupled with the organisation of satellite sequences in high order molecular weight tandem arrays in heterochromatic regions, have been put forward as reasons for supposing that this type of DNA is not normally transcribed. We report here that we have looked for and found evidence of transcription of satellite DNA on lampbrush loops in oocytes of the crested newt, Triturus cristatus carnifex.

Animals↗

PubMed2004

Closing the gaps on human chromosome 19 revealed genes with a high density of repetitive tandemly arrayed elements.

The reported human genome sequence includes about 400 gaps of unknown sequence that were not found in the bacterial artificial chromosome (BAC) and cosmid libraries used for sequencing of the genome. These missing sequences correspond to approximately 1% of euchromatic regions of the human genome. Gap filling is a laborious process because it relies on analysis of random clones of numerous genomic BAC or cosmid libraries. In this work we demonstrate that closing the gaps can be accelerated by a selective recombinational capture of missing chromosomal segments in yeast. The use of both methodologies allowed us to close the four remaining gaps on the human chromosome 19. Analysis of the gap sequences revealed that they contain several abnormalities that could result in instability of the sequences in microbe hosts, including large blocks of micro- and minisatellites and a high density of Alu repeats. Sequencing of the gap regions, in both BAC and YAC forms, allowed us to generate a complete sequence of four genes, including the neuronal cell signaling gene SCK1/SLI. The SCK1/SLI gene contains a record number of minisatellites, most of which are polymorphic and transmitted through meiosis following a Mendelian inheritance. In conclusion, the use of the alternative recombinational cloning system in yeast may greatly accelerate work on closing the remaining gaps in the human genome (as well as in other complex genomes) to achieve the goal of annotation of all human genes.

Alu Elements↗

PubMed2004

Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis.

Many plants increase in freezing tolerance in response to low temperature, a process known as cold acclimation. In Arabidopsis, cold acclimation involves action of the CBF cold response pathway. Key components of the pathway include rapid cold-induced expression of three homologous genes encoding transcriptional activators, CBF1, 2 and 3 (also known as DREB1b, c and a, respectively), followed by expression of CBF-targeted genes, the CBF regulon, that increase freezing tolerance. Unlike Arabidopsis, tomato cannot cold acclimate raising the question of whether it has a functional CBF cold response pathway. Here we show that tomato, like Arabidopsis, encodes three CBF homologs, LeCBF1-3 (Lycopersicon esculentum CBF1-3), that are present in tandem array in the genome. Only the tomato LeCBF1 gene, however, was found to be cold-inducible. As is the case for Arabidopsis CBF1-3, transcripts for LeCBF1-3 did accumulate in response to mechanical agitation, but not in response to drought, ABA or high salinity. Constitutive overexpression of LeCBF1 in transgenic Arabidopsis plants induced expression of CBF-targeted genes and increased freezing tolerance indicating that LeCBF1 encodes a functional homolog of the Arabidopsis CBF1-3 proteins. However, constitutive overexpression of either LeCBF1 or AtCBF3 in transgenic tomato plants did not increase freezing tolerance. Gene expression studies, including the use of a cDNA microarray representing approximately 8000 tomato genes, identified only four genes that were induced 2.5-fold or more in the LeCBF1 or AtCBF3 overexpressing plants, three of which were putative members of the tomato CBF regulon as they were also upregulated in response to low temperature. Additional experiments indicated that of eight tomato genes that were likely orthologs of Arabidopsis CBF regulon genes, none were responsive to CBF overexpression in tomato. From these results, we conclude that tomato has a complete CBF cold response pathway, but that the tomato CBF regulon differs from that of Arabidopsis and appears to be considerably smaller and less diverse in function.

Acclimatization↗

PubMed2002

Length and sequence heterogeneity in 5S rDNA of Populus deltoides.

The 5S rRNA genes and their associated non-transcribed spacer (NTS) regions are present as repeat units arranged in tandem arrays in plant genomes. Length heterogeneity in 5S rDNA repeats was previously identified in Populus deltoides and was also observed in the present study. Primers were designed to amplify the 5S rDNA NTS variants from the P. deltoides genome. The PCR-amplified products from the two accessions of P. deltoides (G3 and G48) suggested the presence of length heterogeneity of 5S rDNA units within and among accessions, and the size of the spacers ranged from 385 to 434 bp. Sequence analysis of the non-transcribed spacer (NTS) revealed two distinct classes of 5S rDNA within both accessions: class 1, which contained GAA trinucleotide microsatellite repeats, and class 2, which lacked the repeats. The class 1 spacer shows length variation owing to the microsatellite, with two clones exhibiting 10 GAA repeat units and one clone exhibiting 16 such repeat units. However, distance analysis shows that class 1 spacer sequences are highly similar inter se, yielding nucleotide diversity (pi) estimates that are less than 0.15% of those obtained for class 2 spacers (pi = 0.0183 vs. 0.1433, respectively). The presence of microsatellite in the NTS region leading to variation in spacer length is reported and discussed for the first time in P. deltoides.

Base Sequence↗

PubMed1994

Isolation of human simple repeat loci by hybridization selection.

We have isolated short tandem repeat arrays from the human genome, using a rapid method involving filter hybridization to enrich for tri- or tetranucleotide tandem repeats. About 30% of clones from the enriched library cross-hybridize with probes containing trimeric or tetrameric tandem arrays, facilitating the rapid isolation of large numbers of clones. In an initial analysis of 54 clones, 46 different tandem arrays were identified. Analysis of these tandem repeat loci by PCR showed that 24 were polymorphic in length; substantially higher levels of polymorphism were displayed by the tetrameric repeat loci isolated than by the trimeric repeats. Primary mapping of these loci by linkage analysis showed that they derive from 17 chromosomes, including the X chromosome. We anticipate the use of this strategy for the efficient isolation of tandem repeats from other sources of genomic DNA, including DNA from flow-sorted chromosomes, and from other species.

Base Sequence↗

PubMed1987

The structure and transcription of an element interspersed between tandem arrays of mini-exon donor RNA genes in Trypanosoma brucei.

Messenger RNAs in Trypanosoma brucei brucei have the same 35 bases at the 5' end. These 35 bases are not encoded contiguously in the genome, but are donated from a 140 base RNA (the mini-exon donor RNA). The mini-exon donor RNA (medRNA) is encoded by 1.35 kbp genes that occur in tandem repeats. A DNA element that is associated with mini-exon donor RNA medRNA genes has been identified and characterised by restriction enzyme mapping and partial sequencing. This element (the medRNA gene associated element: MAE) varies in length between 5.5 and 7 kbp. There are between 20 and 40 copies of the element per haploid genome. In clones of genomic DNA MAEs occurred between two medRNA gene arrays and on both sides of a medRNA gene array. The MAEs were always in the same orientation with respect to the medRNA genes. It is proposed that in the genome MAEs are interspersed between tandem arrays of medRNA genes. The transcription of the element has been investigated. Low levels of a 70-80 base transcript derived from a small part of MAE were detected in steady state RNA. Nuclear run off transcript studies indicated that MAEs were transcribed at high levels and that they possibly contain at least one start and stop of transcription.

Animals↗

PubMed1983

Repeated nucleotide sequence arrays in Balbiani ring 1 of Chironomus tentans contain internally nonrepeating and subrepeating elements.

Balbiani rings in Chironomus are large puffs on salivary gland polytene chromosomes that contain functionally related, but nonidentical genes that code for tissue-specific secretory polypeptides. In situ hybridization was used to select a recombinant plasmid (pCtBR1-1) that contained an insert of Chironomus tentans genomic DNA that originated from Balbiani ring 1. Mapping with restriction endonucleases demonstrated that the insert was 385 bp (base pair) and it contained duplicate clusters of certain cleavage sites about 250 bp apart. This repeat was shown to be part of tandem sequence arrays in the genome by hybridization of radioactive pCtBR1-1 to nitrocellulose blots containing limit and partial restriction endonuclease digests of nuclear DNA. Subsequent sequence analysis of the cloned DNA confirmed the presence of one complete copy of a 246-bp repeat comprised of a 114-bp internally nonrepeating segment and a 132-bp segment containing four 33-bp subrepeats. The subrepeats apparently evolved from a simple 9-bp sequence encoding a consensus tripeptide (Lys-Pro-Ser) in which the first two codons (AAA-CCA) were highly conserved at the nucleotide level. Comparisons between intragenic and interspecific (BRb in Chironomus thummi) copies of corresponding sequences revealed that, during the evolution of these tandemly repeated protein-coding sequences, internally nonrepeated segments were highly conserved and most likely became interspersed by variable segments containing subrepeats that arose from reduplication and divergence of 9-bp repeats.

Amino Acid Sequence↗

PubMed1990

Isolation and characterization of a repetitive DNA element from the genome of the human filarial parasite, Brugia malayi.

The genome of the human filarial parasite Brugia malayi contains at least two major repetitive DNA elements. One, referred to as the HhaI family, consists of 10(4)-10(5) tandemly arrayed copies per haploid genome of a monomer of 322 base pairs and does not contain a cleavage site for the restriction endonuclease MboI. We constructed a library of MboI-digested genomic B. malayi DNA in BamHI-cut M13mp18 resulting in the exclusion of the HhaI repeat family from the library. Hybridization of this genomic library with nick-translated genomic DNA yielded several copies of a repeat family which we have named the BmMboI family. From sequence analysis of more than 50 monomers, which differ from each other in sequence and length, we have been able to divide the monomers into several regions based on the level of sequence conservation. Southern blot analyses of B. malayi genomic DNA digested with a variety of restriction endonucleases and probed with the isolated repeat demonstrate multiple bands of varying sizes except with HindIII-cut DNA, where the repeat is found only in very high-molecular-weight DNA.

Animals↗

PubMed1998

Human megasatellite DNA RS447: copy-number polymorphisms and interspecies conservation.

We previously isolated a novel 4.7-kb RS447 sequence, which tandemly repeated approximately 50-70 copies and resided on human chromosome 4p15 (M. Kogi et al., 1997, Genomics 42: 278-283). Another tandem array (or arrays) of several RS447 copies was hereby identified on the distal part of chromosome 8p. To analyze copy-number polymorphisms of the RS447 repeats, genomic DNA samples of eight nonkindred Japanese were subjected to pulsed-field gel electrophoresis. The copy numbers of the RS447 tandem arrays on 4p15 varied drastically from allele to allele and ranged from approximately 34 to 94 copies. All eight Japanese subjects were apparently heterozygous for the RS447 copy number, and 12 copy-number-different alleles have been at least clearly distinguished. The RS447 tandem repeats were thus found to be hypervariable and highly polymorphic in a human population. The RS447 sequences, however, do not appear to be either "selfish" or "junk" DNA. The unit size and sequence of RS447 were found to be very similar between members in the human genome. The unit size of 4746 bp comprises a putative open reading frame of 1590 bp. The RS447 sequence was well conserved in all the tested mammalian species. The head-to-tail tandem repetitive structure in the RS447 homologs was also confirmed in those species. The RS447 sequence is, therefore, considered to consist of a new class of tandemly repeated satellite DNA elements in the mammalian genome, which may thus be called "megasatellite DNA."

Animals↗

PubMed1999

Rana/Pol III: a family of SINE-like sequences in the genomes of western Palearctic water frogs.

The highly repetitive Rana/Pol III family consists of short, tandemly arrayed sequences, scattered throughout the genomes of Palearctic green water frogs. The repeat unit is about 250 bp in length and is a composite element: it contains a SINE-like retroposon with a tRNA structure, flanked by two short direct repeats, and the occurrence of two internal repeats gives evidence that an additional transposition event may have inserted a segment within the already transposed element. Rana/Pol III family is present in the genomes of Rana lessonae, R. ridibunda, and their hybrid form R. esculenta, as well as in R. shqiperica. R. epeirotica, R. cretensis, and the Italian taxon. These sequences are also present in the Iberian R. perezi, although less abundant, but appear to be lacking in the north African species R. saharica. The distribution of Rana/Pol III in the genomes of Palearctic green frogs is in agreement with the phyletic history based on genetic data. The evolutionary pattern proposed for the genus Rana enables us to suppose that the hybridogenetic mechanism is one of the factors accounting for the possible horizontal transfer of Rana/Pol III elements from the central-north Europe species to R. perezi.

Africa, Northern↗

PubMed1996

Stress-induced rearrangement of Fusarium retrotransposon sequences.

Rearrangement of fusarium oxysporum retrotransposon skippy was induced by growth in the presence of potassium chlorate. Three fungal strains, one sensitive to chlorate (Co60) and two resistant to chlorate and deficient for nitrate reductase (Co65 and Co94), were studied by Southern analysis of their genomic DNA. Polymorphism was detected in their hybridization banding pattern, relative to the wild type grown in the absence of chlorate, using various enzymes with or without restriction sites within the retrotransposon. Results were consistent with the assumption that three different events had occurred in strain Co60: genomic amplification of skippy yielding tandem arrays of the element, generation of new skippy sequences, and deletion of skippy sequences. Amplification of Co60 genomic DNA using the polymerase chain reaction and divergent primers derived from the retrotransposon generated a new band, corresponding to one long terminal repeat plus flanking sequences, that was not present in the wild-type strain. Molecular analysis of nitrate reductase-deficient mutants showed that generation and deletion of skippy sequences, but not genomic amplification in tandem repeats, had occurred in their genomes.

Blotting, Southern↗

PubMed1983

Synthesis of multimeric polyoma virus DNA in mouse L-cells: role of the tsA1S9 gene product.

Several different forms of progeny viral DNA can be identified in polyoma virus (Py)-infected mouse L-cells. The majority comprise mature form I superhelical DNA and the circular, double-stranded "theta" replicating intermediates in which the progeny DNA strands never exceed the unit genome length of the template. There is formed, in addition, a minority fraction of multimeric, linear, double-stranded Py DNA molecules that sediment heterogeneously at 28 to 35S and greater than 35S. Restriction enzyme analysis of these large Py DNA molecules reveals them to be tandem arrays of multiple unit genome lengths, covalently linked head to tail. It is estimated that the 28 to 35S multimeric DNA has an average size of about 20 megadaltons, made up of 6 to 20 Py genome units. The greater than 35S Py DNA is, of course, larger. Kinetic analysis indicates that formation of the monomeric progeny viral DNA and the 28 to 35S multimeric Py DNA reaches a peak at about 35 to 36 h postinfection. Synthesis of the very large linear molecules of greater than 35S is first detected after this interval and continues thereafter. The de novo synthesis of all of these progeny Py DNA molecules proceeds apparently normally in Py-infected tsA1S9 mouse L-cells incubated at 38.5 degrees C under conditions which restrict normal cellular DNA replication. These findings suggest that the cellular DNA topoisomerase II activity, encoded in the tsA1S9 locus (R. W. Colwill and R. Sheinin, submitted for publication), is not required for de novo formation of any form of Py DNA. However, the total amount made and the rate of synthesis of the large molecular weight Py DNA are affected very late in temperature-inactivated tsA1S9 cells.

Animals↗