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.     

Restriction fragment length polymorphism (RFLP) analysis in wheat. I. Genomic DNA library construction and RFLP analysis in common wheat

To develop detailed linkage maps of restriction fragment length polymorphism (RFLP) sites in wheat chromosomes, it was necessary to construct a genomic DNA library and to characterize the clones obtained. Forty-nine per cent of the clones were of single or low copy number per genome. With 91 clones of this class, as probes, and with two to four restriction endonucleases, for DNA digestion, RFLPs were examined among eight common wheats and a single emmer wheat. About 20% of the probes, and 13% of the probe-enzyme combinations revealed genetic polymorphism among the common wheats. DNA deletions account for most of the genetic differences among these wheat genomes. Based on the RFLP data, phylogenetic distances among the nine polyploid wheats were estimated, and a dendrogram showing the genetic relationships among them was constructed.     

Development of a chromosomal arm map for wheat based on RFLP markers

Summary A chromosomal arm map has been developed for common wheat (Triticum aestivum L. em. Thell.) using aneuploid stocks to locate more than 800 restriction fragments corresponding to 210 low-copy DNA clones from barley cDNA, oat cDNA, and wheat genomic libraries. The number of restriction fragments per chromosome arm correlates moderately well with relative DNA content and length of somatic chromosomes. The chromosomal arm locations of loci detected with 6 different clones support an earlier hypothesis for the occurrence of a two-step translocation (4AL to 5AL, 5AL to 7BS, and 7BS to 4AL) in the ancestral wheat genomes. In addition, 1 clone revealed the presence of a 5AL segment translocated to 4AL. Anomalies in aneuploid stocks were also observed and can be explained by intrahomoeologous recombination and polymorphisms among the stocks. We view the development of this chromosomal arm map as a complement to, rather than as a substitute for, a conventional RFLP linkage map in wheat.Paper No. 802 of the Cornell Plant Breeding Series     

rRNA gene organization in the Lyme disease spirochete, Borrelia burgdorferi.

Lyme disease is the most common vector-borne disease in the United States. The causative agent is the spirochete Borrelia burgdorferi. The copy number and organization of the genes encoding the rRNAs of this organism were determined. There is a single gene for 16S rRNA and two copies each of the 23S rRNA and 5S rRNA genes. All of the genes are located within a chromosomal fragment of approximately 9.5 to 10.0 kb. The 23S and 5S rRNA genes are tandemly duplicated in the order 23S-5S-23S-5S and are apparently not linked to the 16S rRNA gene, which is situated over 2 kb upstream from the 23S-5S duplication. The individual copies of the 23S-5S duplication are separated by a 182-bp spacer. Within each 23S-5S unit, an identical 22-bp spacer separates the 23S and 5S rRNA sequences from each other. The genome organization of the 23S-5S gene cluster in a number of different B. burgdorferi isolates obtained at a number of different geographical locations, as well as in several other species of Borrelia, was investigated. All isolates of B. burgdorferi tested displayed the tandem duplication, whereas the closely related species B. hermsii, B. anserina, and B. turicatae all contained a single copy of each of the genes. In addition, different geographical isolates of B. burgdorferi can be differentiated on the basis of a restriction fragment length polymorphism associated with the 23S-5S gene cluster. This polymorphism can be a useful tool for the determination of genetic relatedness between different isolates of B. burgdorferi.     

Development of a fluorophore-ribosomal DNA restriction typing method for monitoring structural shifts of microbial communities

DNA restriction fragment polymorphism technologies such as amplified ribosomal DNA restriction analysis (ARDRA) and terminal restriction fragment length polymorphism (T-RFLP) have been widely used in investigating microbial community structures. However, these methods are limited due to either the low resolution or sensitivity. In this study, a fluorophore-ribosomal DNA restriction typing (f-DRT) approach is developed for structural profiling of microbial communities. 16S rRNA genes are amplified from the community DNA and digested by a single restriction enzyme Msp I. All restriction fragments are end-labeled with a fluorescent nucleotide Cy5-dCTP via a one-step extension reaction and detected with an automated DNA sequencer. All 50 predicted restriction fragments between 100 and 600 bp were detected when twelve single 16S rRNA gene sequences were analyzed using f-DRT approach; 92% of these fragments were determined with accuracy of ±2 bp. In the defined model communities containing five components with different ratios, relative abundance of each component was correctly revealed by this method. The f-DRT analysis also showed structural shifts of intestinal microbiota in carcinogen-treated rats during the formation of precancerous lesions in the colon, as sensitive as multiple digestion-based T-RFLP analysis. This study provides a labor and cost-saving new method for monitoring structural shifts of microbial communities.     

Estimation of 16S rRNA gene copy number in several probiotic Lactobacillus strains isolated from the gastrointestinal tract of chicken

The copy numbers of 16S rRNA genes in 12 probiotic Lactobacillus strains of poultry origin were analyzed. Genomic DNA of the strains was digested with restriction endonucleases that do not cut within the 16S rRNA gene of the strains. This was followed by Southern hybridization with a biotinylated probe complementary to the 16S rRNA gene. The copy number of the 16S rRNA gene within a Lactobacillus species was found to be conserved. From the hybridization results, Lactobacillus salivarius I 24 was estimated to have seven copies of the 16S rRNA gene, Lactobacillus panis C 17 to have five copies and Lactobacillus gallinarum strains I 16 and I 26 four copies. The 16S rRNA gene copy numbers of L. gallinarum and L. panis reported in the present study are the first record. Lactobacillus brevis strains I 12, I 23, I 25, I 211, I 218 and Lactobacillus reuteri strains C 1, C 10, C 16 were estimated to have at least four copies of the 16S rRNA gene. In addition, distinct rRNA restriction patterns which could discriminate the strains of L. reuteri and L. gallinarum were also detected. Information on 16S rRNA gene copy number is important for physiological, evolutionary and population studies of the bacteria.     

Construction of hybrid plasmids containing the yeast replicator

In Saccharomyces cerevisiae strain 6-1G-P188 about 10 per cent of rRNA genes exist as extrachromosomal copies of rDNA repeating units. These extrachromosomal copies can be isolated as covalently closed molecules with lengths around 3mu. We have constructed a set of hybrid plasmids containing the bacterial vector pBR325, the LEU2 gene of yeast encoding beta-isopropylmalatedehydrogenase and various EcoRI restriction fragments of the 3mu DNA. We have tested the ability of our hybrid plasmids to transform LEU2 strain DC5 to leucine prototrophy. One of the plasmids Rcp21/11 transforms DC5 at the frequency comparable with that obtained with YEp13, containing the 2mu DNA replication origin. The 2400 bp EcoRI-B fragment of the 3mu DNA in Rcp21/11 carries a gene for 5S rRNA and two spacers. Our results on transformation experiments allow un to suggest that this EcoRI fragment also carries the 3mu DNA replication origin. Yeast transformants containing this plasmid are highly unstable but during the prolonged growth in selective conditions the stabilization of the LEU+ phenotype is observed being most likely a result of integration of Rcp21/11 into the yeast chromosome.     

Restriction fragment length polymorphisms of 16S rRNA genes in the differentiation of fast-growing mycobacterial species

Abstract DNA from several species of fast growing mycobacteria displayed a characteristic restriction fragment length polymorphism (RFLP) pattern when hybridizated to a Mycobacterium fortuitum 16S rRNA gene fragment. The resulting patterns were identical when comparing different strains belonging to the same species. The RFLP results were consistent with those obtained by DNA-DNA hybridization studies. Using this approach, we have been able to identify the number of copies for 16S rRNA genes in several fast-growing mycobacteria.     

Structure and organization of the 5S rRNA genes (5S DNA) inPinus radiata (Pinaceae)

A 5S rRNA gene (5S DNA) from the coniferPinus radiata D. Don has been cloned and characterized at the nucleotide, genomic and chromosomal levels. Sequencing revealed a repeat unit of 524 base pairs which is present in approximately 3000 copies per diploid genome. Two-dimensional gel electrophoresis indicated that these copies are organized in tandem arrays of various length. Using in situ hybridization techniques, the tandem arrays appear to be present on all of the chromosomes. This complexity of chromosomal organization contrasts markedly with the few sites of uniform length found in angiosperm plants such as wheat, pea, and maize.     

DNA probes with different specificities from a cloned 23S rRNA gene of Micrococcus luteus

A 7500 bp PstI restriction fragment of chromosomal DNA from Micrococcus luteus containing a 23S rRNA gene was cloned in vector pHE3 in E. coli RR 28 (the recombinant plasmid was designated pAR1). A recombinant phage (pAR5) hybridizing to all eubacteria tested was constructed by shotgun subcloning of the PstI fragment in phage M13mp8. Further subcloning of the fragments of the 23S rRNA gene in the vectors pTZ18R and pTZ19R using selected restriction sites of the gene enabled us to select cloned fragments of the 23S rRNA gene representing different specificities. Probes specific for Micrococcus luteus-Micrococcus lylae (pAR28), for the Arthrobacter-Micrococcus group (pAR27), for eubacteria (pAR5), and for the detection of eu- and archaebacteria (the so-called universal probe pAR17) were constructed. The specificity of each probe was analysed by dot hybridization to the chromosomal DNAs of representatives of most of the main phyla of eu- and archaebacteria.     

The isolation of a genomic clone containing the apolipoprotein CII gene and the detection of linkage disequilibrium between two common DNA polymorphisms around the gene

Summary We report the isolation of a genomic clone containing the apolipoprotein CII (apo CII) gene and 5 and 3 flanking sequences. A detailed restriction map of the gene has been constructed and DNA fragments that are unique, or low copy number sequences in the genome have been identified. One of these detects a common restriction fragment length polymorphism (RFLP) with the enzyme BglI. Marked linkage disequilibrium is observed between this RFLP and that detected with the apo CII cDNA clone, even though the two variable restriction enzyme sites are approximately 12 kb apart. However, the usefulness of the apo CII gene as a marker for linkage studies is increased by the use of both RFLPs.     

The identification of Cryptosporidium species and Cryptosporidium parvum directly from whole faeces by analysis of a multiplex PCR of the 18S rRNA gene and by PCR/RFLP of the Cryptosporidium outer wall protein (COWP) gene

A multiplex polymerase chain reaction (PCR) procedure to amplify 18S rRNA gene fragments has been developed. Amplified DNA fragments of the expected size were obtained which were specific for Cryptosporidium parvum and Cryptosporidium wrairi (422 bp), Cryptosporidium baileyi (11106 bp) and Cryptosporidium muris (1346 bp). Criptosporidium parvum and C. wrairi can be distinguished using a PCR/restriction fragment length polymorphism (RFLP) analysis of the Cryptosporidium outer wall protein (COWP) gene, and these two techniques were applied to DNA extracted from whole faeces using a simple and rapid procedure. Cryptosporidium parvum DNA was detected in the faeces of 72 humans and 24 calves where cryptosporidial oocysts were demonstrated using conventional light microscopy. The specific DNA fragments were not amplified using extracts of material containing other lower eukaryotic parasites.     

The ribosomal RNA gene cluster in aneuploid chickens: evidence for increased gene dosage and regulation of gene expression

In the chicken, the nucleolus organizer regions, or sites of the genes encoding 18S, 5.8S, and 28S ribosomal RNA (rRNA), map to one pair of microchromosomes that can be identified by silver nitrate cytochemistry. This nucleolar organizer chromosome also contains the major histocompatibility complex. Chickens aneuploid for this chromosome have been identified and reproduced for over seven generations. Crossing two trisomic parents results in the production of viable disomic, trisomic, and tetrasomic progeny, showing two, three, and four nucleoli and nucleolar organizers per cell, respectively. A molecular analysis of rRNA genes was undertaken to establish the gene copy numbers in the aneuploid genotypes, and to determine if elevated numbers of rRNA genes are stably maintained and inherited over multiple generations. Gene copy numbers were determined using hybridization analysis of erythrocyte DNA obtained from individuals comprising a family which segregated disomic, trisomic, and tetrasomic genotypes. The values obtained were 290, 420, and 570 rDNA repeats per cell for disomic, trisomic, and tetrasomic animals, respectively. These results provide molecular confirmation of the two aneuploid states and show that elevated gene copy numbers have been maintained over multiple generations. Fibroblasts derived from disomic and tetrasomic embryos were found to grow at similar rates in culture, and mature rRNA levels in chicken embryo fibroblasts from disomic, trisomic and tetrasomic embryos were also found to have similar levels of mature rRNA. Therefore, despite the increase in rDNA content, the level of rRNA is regulated to diploid amounts in aneuploid fibroblasts.     

Diversity of thermophilic and non-thermophilic Crenarchaeota at 80 degrees C

A hot spring in the solfataric field of Pisciarelli (Naples-Italy) was analysed for Archaeal diversity. Total DNA was extracted from the environment, archaeal 16S rRNA genes were amplified with Archaea specific primers, and a clone library consisting of 201 clones was established. The clones were grouped in 10 different groups each representing a specific band pattern using restriction fragment length polymorphism (RFLP). Members of all 10 groups were sequenced and phylogenetically analyzed. Surprisingly, a high abundance of clones belonging to non-thermophilic Crenarchaeal clusters were detected together with the thermophilic archaeon Acidianus infernus in this thermophilic environment. Neither Sulfolobus species nor other hyperthermophilic Crenarchaeota were detected in the clone library. The relative abundance of the sequenced clones was confirmed by terminal restriction fragment analyses. Amplification of 16S rRNA genes from Archaea transferred from the surrounding environment was considered negligible because DNA from non-thermophilic Crenarchaeota incubated under conditions similar to the solfatara could not be PCR amplified after 5 min.     

Organization and primary nucleotide sequence of 5S rRNA genes in barley Hordeum vulgare

A BamHI DNA fragment of 301 bp corresponding to the main repeating unit of 5S rRNA was isolated from barley genomic DNA. The primary nucleotide sequence of this fragment was determined and a high level of homology was found between coding sequences of 5S rRNA genes of barley, wheat and rye. At the same time, spacer's nucleotide sequences of different species of cereals were changed dramatically. At least two types of 5S rRNA tandem repeats of 301 and 450 bp were found in barley genome. Polymorphism for restriction fragment length in 5S rRNA repeats allowed to discriminate between all barley varieties used in this work.