Duplication and inversion history of a tandemly repeated genes family.

Given a phylogenetic tree for a family of tandemly repeated genes and their signed order on the chromosome, we aim to find the minimum number of inversions compatible with an evolutionary history of this family. This is the first attempt to account for inversions in an evolutionary model of tandemly repeated genes. We present a branch-and-bound algorithm that finds the exact solution, and a polynomial-time heuristic based on the breakpoint distance. We show, on simulated data, that those algorithms can be used to improve phylogenetic inference of tandemly repeated gene families. An application on a published phylogeny of KRAB zinc finger genes is presented.     

Restriction analysis of tandemly repeated yeast ribosomal RNA genes

Summary Ribosomal RNA (rRNA) genes of Saccharomyces cerevisiae are clustered in a DNA repeat unit of 5.9 megadaltons with the gene order 5S-18S-5.8S-25S rRNA (Nath and Bollon, 1977). By using two restriction endonucleases, EcoRII and HindII, which generate DNA fragments that span contiguous portions of two repeat units, we report that the rRNA gene clusters are tandemly repeated without the intervention of additional spacer DNA.The treatment of yeast DNA with the restriction endonucleases EcoRII and HindII result in the generation of 4 different DNA fragments that are of varying sizes and which hybridize with rRNA. The largest DNA fragments, 3.30 megadaltons in the case of HindII and 3.67 megadaltons in the case of EcoRII, encompass regions that code for the two opposite end regions of the 35S precuursor-rRNA. These two end regions are joined by a constant DNA segment of about 0.9 megadaltons in size of which a 0.08 megadalton segment codes for 5S rRNA. Since the 35S precursor-rRNA includes the 5.8S, 18S and 25S rRNA most of the repeat units containing the 4 rRNA coding genes in yeast are linked to each other contiguously without any intervening spacer DNA.A composite map of the DNA restriction fragments obtained by the action of the restriction endonucleases EcoRI, EcoRII, HindII and HindIII on the 5.9 megadalton repeat unit is presented. Some striking features concerning the location of the restriction sites are noted. Of the total 17 DNA restriction sites present on each repeat unit, 9 are located at or near the 3 transcribed spacer regions contained in the 5 megadalton DNA segment that codes for the 35S precursor-rRNA. The 3 transcribed spacer regions in the 35S precursor-rRNA include the two external transcribed spacer regions and an internal transcribed spacer region, the latter representing the 5.8S rRNA.     

The structure of the human histone genes: clustered but not tandemly repeated

We have isolated five clones containing human histone genes from a human genomic DNA library, using a cloned X. laevis histone H4 cDNA probe (pX1ch4). Each genomic clone has been mapped and the locations of the histone genes have been determined by blot hybridization and hybridization-selection of human histone mRNA. In contrast to the organization of previously characterized histone genes, the human histone genes are clustered in the genome but are not arranged into recognizable repeating units. The extreme lack of organization of the human histone genes may reflect the diminished requirement for rapid synthesis of large quantities of histone proteins during mammalian early development.     

A unique pathway of double-strand break repair operates in tandemly repeated genes.

The RAD52 gene product of the yeast Saccharomyces cerevisiae is required for most spontaneous recombination and almost all double-strand break (DSB) repair. In contrast to recombination elsewhere in the genome, recombination in the ribosomal DNA (rDNA) array is RAD52 independent. To determine the fate of a DSB in the rDNA gene array, a cut site for the HO endonuclease was inserted into the rDNA in a strain containing an inducible HO gene. DSBs were efficiently repaired at this site, even in the absence of the RAD52 gene product. Efficient RAD52-independent DSB repair was also observed at another tandem gene array, CUP1, consisting of 18 repeat units. However, in a smaller CUP1 array, consisting of only three units, most DSBs (ca. 80%) were not repaired and resulted in cell death. All RAD52-independent DSB repair events examined resulted in the loss of one or more repeat units. We propose a model for DSB repair in repeated sequences involving the generation of single-stranded tails followed by reannealing.     

Some extrachromosomal circular DNAs from Drosophila embryos are homologous to tandemly repeated genes

In Drosophila melanogaster embryos we have identified three classes of extrachromosomal circular DNA molecules homologous to the three main families of tandemly repeated genes, 5 S, rDNA and histone. 5 S genes are present in circular multimeric molecules containing up to 16 copies of the 375(±7) base-pair repeated unit. Circular molecules homologous to rDNA are also multimeric molecules, which contain up to ten copies of the 240 base-pair tandemly repeated sequence of the non-transcribed spacer. The two major genomic classes of histone units (4800 and 5000 bases) are found only as monomeric circular molecules.No circular intermediate of the I transposable element was detected in embryos laid by f₁ dysgenic females produced by the I-R system of hybrid dysgenesis.As far as we know, it is the first time that genes have been identified among extrachromosomal circular molecules independently of any specific amplification phenomenon.     

Expression of synthetic calcitonin genes in plasmid vectors containing tandemly repeated non-overlapping ribosome binding sites

1. Expression plasmids containing non-overlapping tandemly repeated ribosome binding sites (RBS) were constructed in order to stabilize mRNA and enhance translation. 2. Two synthetic genes (human calcitonin tetramer gene and a fusion gene human gamma-interferon-human calcitonin) were cloned in these vectors and the effect of multiplicity of Shine-Dalgarno (S/D) sequence on heterologous gene expression was studied. 3. It was found that duplication and triplication of RBS had no effect on the stability of mRNA but led to a strong decrease in the level of recombinant protein and mRNA in the cell. 4. Plasmids bearing four times repeated S/D sequences gave longer-lived mRNAs and maintained a level of protein and mRNA very close to the values obtained with a single S/D containing plasmids.     

A conserved tandemly repeated DNA sequence inCruciferae

Several HindIII monomer units of a tandemly repeated nuclear DNA sequence ofBrassica campestris andBrassica juncea (Cruciferae) have been cloned and sequenced. The monomer units, of 177 bp length, are AT-rich and share 88% homology between themselves and more than 65% homology with similar repeats of otherCruciferae likeBrassica oleracea, Sinapis alba andRaphanus sativus. Thus unlike the rapid divergence of tandemly repeated satellite DNA in other organisms, this DNA element is highly conserved thus indicating its importance.     

Characterization of a tandemly repeated DNA from the fleshflySarcophaga bullata

In studies on the highly repetitive DNA sequences of the flesh flySarcophaga bullata, a 279 bp tandem repeat was cloned and sequenced. A 17 bp stretch within the clone was identical to a motif repeated five times in the satellite DNA of the Bermuda land crab. Southern DNA blotting showed the tandem repeat had a high degree of conservation of MboI sites, but had divergence for EcoRI sites; thus, all repeat units were not identical. The cloned DNA localized to the quinacrine-bright centromeric heterochromatin of the C and E autosomes and to sites on the chromosomal arms. In cases of asynapsis of homologs, the probe localized to euchromatic sites on both homologs or sometimes only on one homolog. The probe also localized near, to, or at a major developmental puff (B9). We conclude that blocks of this short interspersed repetitive DNA occur throughout theSarcophaga genome in both heterochromatin and euchromatin, and also that the variable position of these sequences suggests they possess a degree of instability.     

Cheetahs have 4 serum amyloid a genes evolved through repeated duplication events

Amyloid A (AA) amyloidosis is a leading cause of mortality in captive cheetahs (Acinonyx jubatus). We performed genome walking and PCR cloning and revealed that cheetahs have 4 SAA genes (provisionally named SAA1A, SAA1B, SAA3A, and SAA3B). In addition, we identified multiple nucleotide polymorphisms in the 4 SAA genes by screening 51 cheetahs. The polymorphisms defined 4, 7, 6, and 4 alleles for SAA1A, SAA3A, SAA1B, and SAA3B, respectively. Pedigree analysis of the inheritance of genotypes for the SAA genes revealed that specific combinations of alleles for the 4 SAA genes cosegregated as a unit (haplotype) in pedigrees, indicating that the 4 genes were linked on the same chromosome. Notably, cheetah SAA1A and SAA1B were highly homologous in their nucleotide sequences. Likewise, SAA3A and SAA3B genes were homologous. These observations suggested a model for the evolution of the 4 SAA genes in cheetahs in which duplication of an ancestral SAA gene first gave rise to SAA1 and SAA3. Subsequently, each gene duplicated one more time, uniquely making 4 genes in the cheetah genome. The monomorphism of the cheetah SAA1A protein might be one of the factors responsible for the high incidence of AA amyloidosis in this species.     

Recent evolutionary history of American Onchocerca volvulus, based on analysis of a tandemly repeated DNA sequence family

Polymerase chain reaction (PCR) products were characterized for a repeated sequence family (designated "O-150") of the human filarial parasite Onchocerca volvulus. In phylogenetic inferences, the O-150 sequences clustered into closely related groups, suggesting that concerted evolution maintains sequence homology in this family. Using a novel mathematical model based on a nested application of an analysis of variance, we demonstrated that African rainforest and savannah strain parasite populations are significantly different. In contrast, parasites collected in the New World are indistinguishable from African savannah strains of O. volvulus. This finding supports the hypothesis that onchocerciasis was recently introduced into the New World, possibly as a result of the slave trade.      

Localization of tandemly repeated DMA sequences in Arabidopsis thaliana

In-situ hybridization to interphase nuclei and chromosomes of Arabidopsis thaliana (2n= 10) shows that there are four sites of rDNA in a diploid nucleus. The sites are located on chromosomes 2 and 4, and the strength of hybridization indicates that copy number is similar at both pairs of sites. Hybridization to trisomic line 4 revealed five hybridization sites. Silver staining of nucleoli demonstrates that all four loci can be active in diploid interphase nuclei. The tandemly repeated probe pAL1 hybridizes near to the centromeres of all five chromosome pairs. In diploid interphase nuclei, 10 sites of hybridization are detected, while 15 are seen in triploid nuclei. The sites of hybridization co-localize with the centromeric heterochromatin visualized by staining DNA with the fluorochrome DAPI. The results demonstrate that molecular cytogenetics can be applied to A. thaliana and high resolution physical chromosome maps can be generated. Both probes may be useful for interphase cytogenetics, where they enable chromosome number and aneuploidy to be examined in tissues without divisions. The physical localization of these hybridization sites provides a starting point for linking RFLP and physical chromosome maps.     

In Euglena, spliced-leader RNA (SL-RNA) and 5S rRNA genes are tandemly repeated.

In Euglena gracilis, a 26 nucleotide leader sequence (spliced leader sequence = SL) is transferred by trans-splicing to the 5' end of a vast majority of cytoplasmic mRNAs (8). The SL originates from the 5' extremity of a family of closely related snRNAs (SL-RNAs) which are about 100 nucleotide long. In this paper we present the nucleotide sequences of two SL-RNA genes, confirming the sequences previously established by sequencing purified SL-RNAs. Although some SL-RNA genes are dispersed throughout the genome, we show that the majority of SL-RNA genes are located on 0.6 kb repeated units which also encode the cytoplasmic 5S rRNA. We estimate that the copy number of these repeated units is about 300 per haploid genome. The association of SL-RNA and 5S rRNA genes in tandemly repeated units is also found in nematodes but paradoxically does not exist in trypanosomes which are phylogenically much closer to Euglena. We also show that a high number of sequences analogous to the 26 nucleotide SL are dispersed throughout the genome and are not associated with SL-RNAs.     

Polymorphisms in tandemly repeated sequences ofSaccharomyces cerevisiae mitodhondrial DNA

Summary A spontaneously arising mitochondrial DNA (mtDNA) variant ofSaccharomyces cerevisiae has been formed by two exta copies of a 14-bp sequence (TTAATTAAATTATC) being added to a tandem repeat of this unit. Similar polymorphisms in tandemly repeated sequences have been found in a comparison between mtDNAs from our strain and others. In 5850 bp of intergenic mtDNA squence, polymorphisms in tandemly repeated sequences of three or more base pairs occur approximately every 400–500 bp whereas differences in 1–2 bp occur approximately every 60 bp. Some polymorphisms are associated wit optional G+C-rich sequences (GC clusters). Two such optional GC clusters and one A+T repeat polymorphism have been discovered in the tRNA synthesis locus. In addition, the variable presence of large open reading frames are documented and mechanisms for generating intergenic sequence diversity inS. cerevisiae mtDNA are discussed.