Amplified ribosomal spacer sequence: structure and evolutionary origin

A novel class of repeated sequences consisting of tandem arrays of ribosomal spacer sequence has been discovered in a mouse genome. Comparison to normal ribosomal DNA reveals that one repeat unit consists of two separate parts of spacer sequence. This amplified spacer sequence has a pseudogene-like structure but is distinct from the previously reported pseudogenes and orphons in regions lacking coding sequences. So far the amplified spacer sequence has been found only in the BALB/c mouse genome but not in ten other laboratory strains and several wild-type mouse stocks. Surprisingly, a part of the amplified spacer sequence unit had a higher homology to the corresponding part of the ribosomal DNA sequence of Mus musculus molossinus, a Japanese wild-type mouse, than to the corresponding part of the rDNA of the BALB/c mouse. These findings suggest that the amplified spacer sequence of the BALB/c mouse might have partly originated in M. m. molossinus or in a related subspecies.     

Characterization of mouse ribosomal gene fragments purified by molecular cloning.

Four mouse ribosomal gene fragments cloned in lambda gtWES were studied by restriction enzyme mapping and Southern transfer experiments. These fragments were found to contain 18S DNA and transcribed as well as non-transcribed spacer DNA. Variation in the structure of these mouse DNA inserts was limited to one region of spacer DNA. This variation may reflect real structural differences found in mouse ribosomal genes or possibly deletion events which occurred during cloning. The transcribed regions of the inserts appear identical to one antoher and restriction enzyme fragments from this region correspond to fragments observed in digests of total mouse DNA. These clones will be useful in studying the structure of transcribed spacer DNA including the ribosomal gene promoter.     

Study of polymorphism in human rRNA gene clusters

Human ribosomal DNA (rDNA) probe specific for the 3' end of the 28S rRNA gene was used for detecting standard restriction fragments' length polymorphism (RFLPs) in the non-transcribed spacer. The conditions for hybridization of rDNA probe which eliminate cross hybridization of parts of 28S rRNA gene were developed. A test for detecting incompletely restricted DNA was also developed which may be used in experiments for detecting new RFLPs. It was found that a set of standard RFLPs was identical in various human tissues for one individual. Frequency of standard RFLPs in the non-transcribed spacer of human rRNA gene clusters was calculated.     

Sequences and evolutionary analysis of mouse 5S rDNAs

We selectively amplified the spacer regions of genes for mouse 5S ribosomal RNA (rRNA), which are tandemly repeated, by the PCR method, using primers specific to the two ends of the coding region for 5S rRNA. Fragments of approximately 1.6 kb were amplified from DNA from the BALB/cCrSlc mouse (Mus musculus domesticus), the SM/J mouse (M. m. domesticus), the MOA mouse (M. m. musculus) and the SEG mouse (M. spretus). These fragments were cloned into an appropriate plasmid vector, and two clones representative of each of the four strains were sequenced. The sequences were GC rich (> 60%) and contained a high proportion of very simple repetitive motifs, such as (TG)n and (ATCC)n, which accounted for the intra- and intergenomic length heterogeneity. Excluding such polymorphic regions and neglecting small insertions or deletions, we estimated the sequence divergence between clones. Sequence divergence within a genome averaged 0.26%, and the divergence between individuals of the same subspecies, between subspecies, and between species was 0.44%, 0.62%, and 1.73%, respectively. The results indicate that the spacer region evolved rapidly but with a reduction in heterogeneity within each genome, as a result of certain, as yet unidentified, homogenization mechanisms. The results further suggest that the spacer regions of genes for 5S rRNA may provide good indicators for phylogenetic analysis of closely related species.      

Differentiation of mosquito-pathogenic strains of Bacillus sphaericus from non-toxic varieties by ribosomal RNA gene restriction patterns.

DNA from 17 strains of Bacillus sphaericus, including representatives of all the established DNA homology groups, was cleaved with EcoRI or HindIII and fragments were separated by agarose gel electrophoresis. Southern blots of this DNA were hybridized to a radioactively labelled DNA probe prepared from the cloned 16S rrnB ribosomal RNA operon of Escherichia coli. Banding patterns of the chromosomal DNA digests and the autoradiograms were specific to DNA homology groups I (B. sphaericus sensu stricto), IIA (mosquito-pathogenic strains), IIB (B. fusiformis) and V, but groups III and IV were not clearly distinguished. This suggests that the mosquito-pathogenic strains represent a separate subspecies.     

Genetic polymorphism of the murine myogenic gene Myo-D1.

Polymorphism of the myogenic gene, Myo-D1, has been sought to examine genetic mechanisms which control skeletal muscle development. By Southern analysis, three restriction-fragment length polymorphisms (RFLPs) have been found in various mouse strains using the TaqI, SacI and BglII restriction endonucleases and a full-length cDNA Myo-D1 probe. Reference to the distribution of RFLPs in different mouse strains derived from Mus mus (M.m.) domesticus and M.m. musculus subspecies suggests that Myo-D1 rearrangements are subject to nonrandom association. The biological significance of RFLP of the Myo-D1 gene is yet to be determined.     

Molecular polymorphism and phenotypic variation in Aspergillus carbonarius

Thirteen collection strains and field isolates of Aspergillus carbonarius were examined by using various genotypic and phenotypic approaches. Restriction fragment length polymorphism analysis of the ribosomal RNA gene cluster and the mitochondrial DNA of the strains revealed only slight variations, except for one field isolate (IN7), which exhibited completely different ribosomal RNA gene cluster and mitochondrial DNA patterns. The mitochondrial DNAs of these strains were found to be much larger (45 to 57 kb) than those found earlier in the A. niger aggregate. Strain-specific characters could be detected by the random amplified polymorphic DNA technique. Isoenzyme analysis and examination of carbon source utilisation patterns of the strains also revealed some intraspecific variability, though much smaller than that observed by using DNA-based techniques. The dendrograms constructed based on genotypic and phenotypic data suggest that strain IN7 might represent a new subspecies of A. carbonarius.Abbreviations kb kilobase pair - mtDNA mitochondrial DNA - RAPD random amplified polymorphic DNA - rDNA ribosomal RNA gene cluster - RFLP restriction fragment length polymorphisms     

Mouse phenome research: implications of genetic background

Now that sequencing of the mouse genome has been completed, the function of each gene remains to be elucidated through phenotypic analysis. The "genetic background" (in which each gene functions) is defined as the genotype of all other related genes that may interact with the gene of interest, and therefore potentially influences the specific phenotype. To understand the nature and importance of genetic background on phenotypic expression of specific genes, it is necessary to know the origin and evolutionary history of the laboratory mouse genome. Molecular analysis has indicated that the fancy mice of Japan and Europe contributed significantly to the origin of today's laboratory mice. The genetic background of present-day laboratory mice varies by mouse strain, but is mainly derived from the European domesticus subspecies group and to a lesser degree from Asian mice, probably Japanese fancy mice, which belong to the musculus subspecies group. Inbred laboratory mouse strains are genetically uniform due to extensive inbreeding, and they have greatly contributed to the genetic analysis of many Mendelian traits. Meanwhile, for a variety of practical reasons, many transgenic and targeted mutant mice have been created in mice of mixed genetic backgrounds to elucidate the function of the genes, although efforts have been made to create inbred transgenic mice and targeted mutant mice with coisogenic embryonic stem cell lines. Inbred mouse strains have provided uniform genetic background for accurate evaluation of specific genes phenotypes, thus eliminating the phenotypic variations caused by mixed genetic backgrounds. However, the process of inbreeding and selection of various inbred strain characteristics has resulted in inadvertent selection of other undesirable genetic characteristics and mutations that may influence the genotype and preclude effective phenotypic analysis. Because many of the common inbred mouse stains have been established from relatively small gene pools, common inbred strains have limitations in their genetic polymorphisms and phenotypic variations. Wild-derived mouse strains can complement deficiencies of common inbred mouse strains, providing novel allelic variants and phenotypes. Although wild-derived strains are not as tame as the common laboratory strains, their genetic characteristics are attractive for the future study of gene function.     

Evolutionary relationships between laboratory mice and subspecies ofMus musculus based on the restriction fragment length variants of the chymotrypsin gene at thePrt-2 locus

Restriction endonuclease fragment length variants in mice were compared by Southern blot analysis using the cDNA probe pcXP33 for the chymotrypsin gene. The variants were detected in the restriction patterns generated by fragments from digestions withBglII,EcoRI,HindIII,Pstl,SacI, andXbaI. The set of protein phenotypes and the restriction patterns of chymotrypsin gene were examined in many laboratory strains and wild subspecies. Most laboratory strains (26 strains) are grouped into a set defined as Set 1, but only a few laboratory strains (AU/SsJ and five BALB/c sublines) are classified as belonging to Set 2. Of wild subspecies, only BRV-MPL (M. brevirostris) can be placed in Set 1, while DOM-BLG and SK/Cam (M. domesticus) belong in Set 2. The assignment of an appropriate set defined by the characteristics of the chymotrypsin gene has also been investigated inM. musculus, two Chinese subspecies,M. yamashinai, M. molossinus, andM. castaneus, and the evolutionary relationship between laboratory mice and various subspecies ofMus has been examined.     

Differentiation of lactococci by rRNA gene restriction analysis

Strains of the subspecies of Lactococcus lactis could be differentiated by rRNA gene restriction fragment length polymorphisms (RFLP). 16S rRNA-specific oligonucleotide as well as polynucleotide DNA probes were used for the detection of restriction fragments. In addition, a site-specific probe was designed for the intergenic spacer region of 23S and 5S rRNA genes. For all lactococcal strains the putative presence of six rRNA operons was confirmed. A non-radioactive hybridization assay was used based on hybrid detection by chemiluminescence. Specific patterns were found for any of the strains investigated. Subspecies-specific restriction fragments could be identified in addition to the strain-specific patterns.     

The consequence of natural selection on genetic variation in the mouse

Laboratory mouse strains are known to have emerged from recent interbreeding between individuals of Mus musculus isolated populations. As a result of this breeding history, the collection of polymorphisms observed between laboratory mouse strains is likely to harbor the effects of natural selection between reproductively isolated populations. Until now no study has systematically investigated the consequences of this breeding history on gene evolution. Here we have used a novel, unbiased evolutionary approach to predict the founder origin of laboratory mouse strains and to assess the balance between ancient and newly emerged mutations in the founder subspecies. Our results confirm a contribution from at least four distinct subspecies. Additionally, our method allowed us to identify regions of relaxed selective constraint among laboratory mouse strains. This unique structure of variation is likely to have significant consequences on the use of mouse to find genes underlying phenotypic variation.     

Characterization of the 23S and 5S rRNA genes and 23S-5S intergenic spacer region (ITS-2) of Photobacterium damselae

The 23S ribosomal RNA (rRNA) gene has been sequenced in strains of the fish pathogens Photobacterium damselae subsp. damselae (ATCC 33539) and subsp. piscicida (ATCC 29690), showing that 3 nucleotide positions are clearly different between subspecies. In addition, the 5S rRNA gene plus the intergenic spacer region between the 23S and 5S rRNA genes (ITS-2) were amplified, cloned and sequenced for the 2 reference strains as well as the field isolates RG91 (subsp. damselae) and DI21 (subsp. piscicida). A 100% similarity was found for the consensus 5S rRNA gene sequence in the 2 subspecies, although some microheterogeneity was detected as inter-cistronic variability within the same chromosome. Sequence analysis of the spacer region between the 23S and 5S rRNA genes revealed 2 conserved and 3 variable nucleotide sequence blocks, and 4 different modular organizations were found. The ITS-2 spacer region exhibited both inter-subspecies and intercistronic polymorphism, with a mosaic-like structure. The EMBL accession numbers for the 23S, 5S and ITS-2 sequences are: P. damselae subsp. piscicida 5S gene (AJ274379), P. damselae subsp. damselae 23S gene (Y18520), subsp. piscicida 23S gene (Y17901), P. damselae subsp. piscicida ITS-2 (AJ250695, AJ250696), P. damselae subsp. damselae ITS-2 (AJ250697, AJ250698).     

A molecular basis for discrete size variation in human ribosomal DNA.

The tandemly repeated human ribosomal RNA (rRNA) genes contain a region of size heterogeneity that is present in the nontranscribed spacer of every individual examined. This heterogeneity has been previously examined by Southern analysis of BamHI-digested human DNA. Using a ribosomal DNA (rDNA) probe specific for the 3' end of the 28S rRNA gene, at least four discrete sizes of BamHI fragments were seen in human populations. Molecular analysis of the cloned DNA from this region reveals tandem duplication of a segment of spacer rDNA located 388 base pairs (bp) 3' to the end of the 28S ribosomal RNA gene. Five hundred fifty bp of DNA, flanked on either side by a 150-bp repeated element, is either duplicated or deleted to produce a series of spacers that differ in size by 850 bp. These duplications/deletions appear to be the product of unequal homologous exchange, mediated by the small repeated element. Thus, human rDNA fragments cloned in lambda vectors and propagated in E. coli generate the same apparent size variation seen in genomic DNA. This study suggests that unequal homologous exchange is the molecular basis for the observed length heterogeneity in the spacer rDNA and may be a common mechanism for the generation of human genetic diversity.     

Cloning and characterization of the ribosomal RNA gene complex from the plant pathogen Verticillium albo-atrum

Abstract The DNA coding for the ribosomal RNA gene complex (rDNA) has been cloned from isolate 621P(PV1) of Verticillium albo-atrum which is pathogenic for hops ( Humulus lupulus ). The rDNA was mapped using a range of restriction enzymes. The functional units of the intergenic spacer (IGS), 18S, 5.8S and 25S regions were located by hybridization to specific gene probes from the rDNA complex of Aspergillus nidulans . The start points of the 18S and 5.8S regions were confirmed by partial sequencing. A genomic restriction enzyme map was found to be identical with the map of the cloned DNA. The rDNA repeat was 7.6 kb in length and this was used as an homologous probe to analyse the size of the repeat in 18 hop isolates of V. albo-atrum strains and in one isolate from alfalfa (Luc2). All of the isolates had a repeat size of 7.6 kb except for Luc2 where the rDNA complex was 8.4 kb.     

Ribosomal cistrons in Bothrops neuwiedi (Serpentes) subspecies from Brazil.

The ribosomal cistrons of six subspecies of Bothrops neuwiedi (Serpentes) were studied at both the cytogenetic and molecular levels. These subspecies populations occur in several Brazilian regions. The analysis of the nucleolar organizing region banding patterns showed variability in the chromosomal localization of rDNA cistrons. The rDNA clusters were found in two microchromosomes, in chromosome pair Number 6, or even in the one homologue of chromosome 6, and one microchromosome, according to the specimen examined but independent of the population from which it was selected. The organization of the rDNA repeat was studied by Southern blot hybridization using Xenopus laevis ribosomal DNA probes. The size of the repeat is 10.4 kilobases (kb), and the intergenic spacer (IGS), 2.6 kb long, is relatively small compared with the size of other vertebrate IGSs. Restriction mapping using the restriction enzymes EcoRI, BamHI, HindIII, and PvuII showed a highly conserved organization of the ribosomal repeats in the total genomic DNA of the subspecies studied.     

A member of a new repeated sequence family which is conserved throughout eucaryotic evolution is found between the human delta and beta globin genes

A new class of human interspersed repeated sequences distinct from the AluI family was found by screening a human gene library with a mouse ribosomal gene non-transcribed spacer probe (rDNA NTS). A member of this sequence family was localized to a 251 bp segment between the human delta and beta globin genes: a region previously judged to be devoid of repeated DNA. The complete nucleotide sequence of this segment revealed a tandem block of 17 TG dinucleotides, a feature hypothesized by others to be a recombination hot spot responsible for gene conversion in the gamma globin locus region. When the genomes of Xenopus, pigeon, slime mold and yeast were examined, reiterated sequences homologous to both the mouse rDNA NTS and human globin repeat were found in every case. The discovery of this extraordinarily conserved repeated sequence family appears to have depended upon not using salmon sperm DNA during hybridization. The use of eucaryotic carrier DNA may bias the search for repeated sequences against any which may be highly conserved during eucaryotic evolution.