Analysis of repetitive sequence elements containing tRNA-like sequences.

Several repetitive sequence elements from diverse species share extensive sequence homology with tRNA molecules. Analysis of the tRNA-like sequences within these elements suggest that they have originated from authentic tRNA sequences. Elements containing tRNA-like sequences can be divided into three distinct groups whose members share extensive sequence homology, have similar sequence organization and have unique species distribution. We suggest that these three groups represent independent examples of retroposon families that have originated from tRNAs.     

Activation of insulin-like growth factor II expression during skeletal muscle regeneration in the rat: correlation with myotube formation.

Insulin-like growth factors (IGFs) are important stimulators of proliferation and differentiation of cultured myoblasts. It has previously been shown that IGF-I is induced during muscle regeneration in rodents, however, little is known about the expression of IGF-II. Therefore, two in vivo models were used to analyze IGF-II mRNA expression during skeletal muscle regeneration in the rat: injection of the snake venom notexin and induction of ischemia. During the regeneration process the levels of both IGF-I and IGF-II mRNA were transiently induced, as analyzed by solution hybridization. Both IGF-I-like immunoreactivity and IGF-II-like immunoreactivity were found to be present during muscle regeneration. In a time course study, induction of IGF-II was preceded by IGF-I, both at the mRNA and protein levels. Using alpha- and beta-actin as markers for different stages of skeletal muscle differentiation, together with the immunohistochemistry data, it is concluded that the expression of IGF-I and IGF-II occurs at different differentiation stages, and that IGF-II appears concomitant to the formation of myotubes. These results suggest that each IGF has a distinct role during the differentiation of muscle cells.     

Transformation-defective v-ski induces MyoD and myogenin expression but not myotube formation.

The ski oncogene induces muscle differentiation in otherwise nonmyogenic quail embryo cells (C. Colmenares and E. Stavnezer, Cell 59:293-303, 1989). Here we report that v-ski induces both MyoD and myogenin expression, suggesting that activation of these muscle regulatory genes may be a critical step in ski-induced myogenesis. We also describe a transformation-defective mutant of v-ski (tdM5i) that fails to induce myotube formation, although it induces the expression of many muscle-specific genes, including the MyoD and myogenin genes. Therefore, if activation of MyoD and myogenin expression is a necessary component of the myogenic program triggered by ski, it is clearly insufficient to account for complete muscle differentiation.     

Preferential expression of unique sequences adjacent to middle repetitive sequences in mouse cytoplasmic RNA.

Total single-copy DNA and single-copy DNA contiguous to middle repetitive sequences were isolated from mouse brain by successive hydroxylapatite column chromatographies. These DNAs, termed repeat-contiguous single-copy DNA, were found to constitute 48% of the total single-copy DNA. The saturation hybridization values of these two DNA probes to nuclear RNA and cytoplasmic RNA containing polyA of mouse brain and liver were measured. The saturation hybridization levels of total single-copy DNA to brain and liver nuclear RNA were 13.5% and 8.8%, respectively, and those of repeat-contiguous single-copy DNA to the same RNA samples were 13.3% and 8.5%, respectively. On the contrary, the saturation hybridization levels of single-copy DNA to cytoplasmic RNA containing polyA of brain and liver were 3.8% and 2.0%, respectively, and those of repeat-contiguous single-copy DNA to the same RNA samples were 5.8% and 4.0%, respectively. Similar results were obtained with total cytoplasmic RNA. These results indicate that about half the steady state nuclear RNA is transcribed from repeat-contiguous single-copy DNA, and that cytoplasmic RNA containing polyA is mainly derived from repeat-contiguous single-copy DNA.     

Clustered and interspersed repetitive DNA sequence family of Chironomus. The nucleotide sequence of the Cla-elements and of various flanking sequences

The nucleotide sequence of more than 30 cloned members of the clustered and interspersed repetitive Cla-sequence family present in the genome of various chironomids has been determined. In four cloned Cla-element clusters, the 5' and 3'-flanking sequences including the junctions between the Cla-element clusters and the flanking sequences were also sequenced. The repetitive Cla-elements, which are able to transpose under certain circumstances, have a monomer length ranging from 110 to 119 base-pairs, are very A + T-rich (greater than 80% A + T) and display numerous palindromic sequences. The Cla-elements are organized in small (4 elements) to medium-sized (greater than 30 elements) tandem repetitive clusters, which are dispersed over more than 200 sites of the chromosomes of Chironomus thummi thummi, including the non-transcribed spacer of the ribosomal DNA repeating unit. The tandem repetitive Cla-elements show anomalous behaviour during high-percentage polyacrylamide gel electrophoresis, indicating a bent or globular conformation. The flanking sequences are also repetitive, but the sequenced parts did not reveal any tandem repetitive arrangement. Near the junctions of the Cla-element clusters and the flanking sequences, short duplications are found, ranging from 5 to 12 bases, present in both sides of the Cla-element clusters. The Cla-elements might be involved in the hybrid dysgenesis phenomenon that is observed after crossings between the two subspecies Ch. th. thummi and Ch. th. piger.     

Methylation of repetitive DNA sequences in the brain during aging of the rat

Methylation of repetitive DNA sequences (RDS) of the genomic DNA of the brain of 15- and 88-week old rats was analysed by digestion with HpaII, MspI, EcoRI + HpaII and EcoRI + MspI followed by end-labelling. mCpG doublets are present in two RDS of approximately 5 and 0.4 kb, and are also randomly distributed throughout the genome. Hemimethylated mCpC doublets also occur. Both mCpG and mCpC doublets are found more in the old than in the young. This age-related increase in DNA methylation occurs both at CCGG sites of the RDS and in the entire genome. Such increase in DNA methylation may alter chromatin conformation and gene expression in the brain as the rat ages.     

Analysis of rat repetitive DNA sequences.

Parameters of repetitive sequence organization have been measured in the rat genome. Experiments using melting, hydroxylapatite binding, and single strand specific nuclease digestion have been used to measure the number, length, and arrangement of repeated DNA sequences. Renaturation and melting or S1 nuclease digestion of 1.0 kbp DNA fragment show about 20% of rat DNA sequences are 3000-fold repeated. Renatured duplexes from 4.0 kbp DNA fragments display two repetitive size fractions after nuclease digestion. About 60% of the repeated sequences are 0.2-0.4 kbp long while the remainder are longer than 1.5 kbp. The arrangement of the repeated sequences has been measured by hydroxylapatite fractionation of DNA fragments of varying lengths bearing a repeated sequence. Repeated DNA sequences are interspersed among 2.5 kbp long nonrepeated sequences throughout more than 70% of the rat genome. There are approximately 350 different 3000-fold short repeated sequences in the rat interspersed among 600,000 nonrepeated DNA sequences.     

Highly repetitive DNA sequences in cyanobacterial genomes.

We characterized three distinct families of repeated sequences in the genome of the cyanobacterium Calothrix sp. strain PCC 7601. These repeated sequences were present at a level of about 100 copies per Calothrix genome and consisted of tandemly amplified heptanucleotides. These elements were named short tandemly repeated repetitive (STRR) sequences. We used the three different Calothrix STRR sequences as probes to perform Southern hybridization experiments with DNAs extracted from various cyanobacterial strains, Bacillus subtilis, and Escherichia coli. The three different STRR sequences were found as repetitive genomic DNA components specific to the heterocystous strains tested. The role of the STRR sequences, as well as their possible use in taxonomic studies, is discussed.     

Medium reiteration frequency repetitive sequences in the human genome.

Fourteen novel medium reiteration frequency (MER) families were found, in the human genome, by using two different methods. Repetition frequencies per haploid human genome were estimated for each of these families as well as for six previously described MER DNA families. By these measurements, the families were found to contain variable numbers of elements, ranging from 200 to 10,000 copies per haploid human genome.     

Evolutionary trends of different repetitive DNA sequences during speciation in the genus secale

The presence and distribution of two simple sequence repeats (SSRs), three highly repetitive sequences from rye, and the 5S rDNA have been investigated in 3 rye cultivars and 10 wild-related species of the genus SECALE: The following conclusions can be drawn in addition to detailed knowledge of the sequence content of chromatin in each accession studied: (1) Every species is unique in either or both the complement and chromosomal distribution of the six repeated sequences analyzed. (2) These sequences reveal multiple landmarks along all the rye chromosomes arms. (3) High polymorphism as well as heterozygosity between homologues in the distribution of the (AAG)(5) and (AAC)(5) was revealed in the outbreeding species of the Secale strictum complex. (4) It is possible to deduce trends in the complexity of repetitive DNA during the evolution of the genus. A possible evolutionary pathway that accounts for the present-day Secale species is presented.     

Arrangement of repetitive sequences in the genome of herpesvirus Sylvilagus.

Herpesvirus sylvilagus is a lymphotropic (type gamma) herpesvirus of cottontail rabbits (Sylvilagus floridanus). Analysis of virion DNA of herpesvirus sylvilagus has revealed that the genome consists of one stretch of about 120 kilobase pairs of internal, unique DNA flanked by a variable number of 553-base-pair tandem repeats. The G + C content of the repetitive DNA is extremely high (83%), as determined by sequencing. The organization of the herpesvirus sylvilagus genome is, therefore, similar to that of the primate lymphotropic viruses herpesvirus saimiri and herpesvirus ateles.     

Genome-specific repetitive sequences in the genus Oryza

Summary Repetitive DNA sequences are useful molecular markers for studying plant genome evolution and species divergence. In this paper, we report the isolation and characterization of four genome-type specific repetitive DNA sequences in the genus Oryza. Sequences specific to the AA, CC, EE or FF genome types are described. These genome-type specific repetitive sequences will be useful in classifying unknown species of wild or domestic rice, and in studying genome evolution at the molecular level. Using an AA genome-specific repetitive DNA sequence (pOs48) as a hybridization probe, considerable differences in its copy number were found among different varieties of Asian-cultivated rice (O. sativa) and other related species within the AA genome type. Thus, the relationship among some of the members of AA genome type can be deduced based on the degree of DNA sequence similarity of this repetitive sequence.     

Optimization of bovine satellite cell-derived myotube formation in vitro

Post-natal myogenic satellite cells, isolated from the sternomandibularis muscles of bovine at slaughter were used for primary culture studies. Isolated satellite cells tended to differentiate into multinucleated myotubes more efficiently if initially plated on to a fibronectin substratum. Bovine-derived satellite cells displayed greater fused cell numbers when exposed to Dulbecco's Modified Eagle's Medium (DMEM) supplemented with horse serum than similar supplementation with fetal calf serum (P less than 0.05) or sheep serum (P less than 0.05). In addition, differentiation appeared nearly complete after 4 days exposure to DMEM-1% horse serum as verified by beta-D-arabinofuranosyl-cytosine addition to cultures. Collectively, these data provide the first evidence that satellite cells can be isolated from a bovine skeletal muscle. Furthermore, these data indicate that bovine-derived satellite cells can be induced to undergo substantial morphological differentiation in vitro.     

Differential roles of HIC-5 isoforms in the regulation of cell death and myotube formation during myogenesis

Hic-5 is a LIM-Only member of the paxillin superfamily of focal adhesion proteins. It has been shown to regulate a range of biological processes including: senescence, tumorigenesis, steroid hormone action, integrin signaling, differentiation, and apoptosis. To better understand the roles of Hic-5 during development, we initiated a detailed analysis of Hic-5 expression and function in C(2)C(12) myoblasts, a well-established model for myogenesis. We have found that: (1) myoblasts express at least 6 distinct Hic-5 isoforms; (2) the two predominant isoforms, Hic-5alpha and Hic-5beta, are differentially expressed during myogenesis; (3) any experimentally induced change in Hic-5 expression results in a substantial increase in apoptosis during differentiation; (4) ectopic expression of Hic-5alpha is permissive to differentiation while expression of either Hic-5beta or antisense Hic-5 blocks myoblast fusion but not chemodifferentiation; (5) Hic-5 localizes to focal adhesions in C(2)C(12) myoblasts and perturbation of Hic-5 leads to defects in cell spreading; (6) alterations in Hic-5 expression interfere with the normal dynamics of laminin expression; and (7) ectopic laminin, but not fibronectin, can rescue the Hic-5-induced blockade of myoblast survival and differentiation. Our data demonstrate differential roles for individual Hic-5 isoforms during myogenesis and support the hypothesis that Hic-5 mediates these effects via integrin signaling.     

Simple repetitive sequences in the genomes of archaebacteria

Stretches of simple sequences poly(dG-dT).poly(dC-dA), poly(dG-dA).poly(dC-dT), poly(dG).poly(dC) and poly(dA).poly(dT), the occurrence of which is a characteristic feature of eukaryotic genomes, are found in the genomes of archaebacteria Halobacterium halobium and Sulfolobus acidocaldarius. In S. acidocaldarius these sequences constitute a considerable portion of the genome; they belong to a class of repetitive sequences dispersed throughout the genome, being transcribed and found in RNAs of different lengths.