A physical and genetic map of the Spiroplasma citri genome.

A physical and genetic map of the Spiroplasma citri genome has been constructed using several restriction enzymes and pulsed field gel electrophoresis. A number of genes were subsequently localized on the map by the use of appropriate probes. The genome size of the spiroplasma estimated from restriction fragments is close to 1780 kbp, the largest of all Mollicutes studied so far. It contains multisite insertions of Spiroplasma virus 1 (SpV1) sequences. The physical and genetic map of the S. citri genome shares several features with that of other Mollicutes, especially those in the Mycoplasma mycoides cluster. This supports the finding that S. citri and these Mycoplasma spp. are phylogenetically related.     

Correlated physical and genetic map of the Bradyrhizobium japonicum 110 genome.

We describe a compilation of 79 known genes of Bradyrhizobium japonicum 110, 63 of which were placed on a correlated physical and genetic map of the chromosome. Genomic DNA was restricted with enzymes PacI, PmeI, and SwaI, which yielded two, five, and nine fragments, respectively. Linkage of some of the fragments was established by performing Southern blot hybridization experiments. For probes we used isolated, labelled fragments that were produced either by PmeI or by SwaI. Genes were mapped on individual restriction fragments by performing gene-directed mutagenesis. The principle of this method was to introduce recognition sites for all three restriction enzymes mentioned above into or very near the desired gene loci. Pulsed-field gel electrophoresis of restricted mutant DNA then resulted in an altered fragment pattern compared with wild-type DNA. This allowed us to identify overlapping fragments and to determine the exact position of any selected gene locus. The technique was limited only by the accuracy of the fragment size estimates. After linkage of all of the restriction fragments we concluded that the B. japonicum genome consists of a single, circular chromosome that is approximately 8,700 kb long. Genes directly concerned with nodulation and symbiotic nitrogen fixation are clustered in a chromosomal section that is about 380 kb long.     

Combined genetic and physical map of the complex genome of Agrobacterium tumefaciens.

A combined genetic and physical map of the Agrobacterium tumefaciens A348 (derivative of C58) genome was constructed to address the discrepancy between initial single-chromosome genetic maps and more recent physical mapping data supporting the presence of two nonhomologous chromosomes. The combined map confirms the two-chromosome genomic structure and the correspondence of the initial genetic maps to the circular chromosome. The linear chromosome is almost devoid of auxotrophic markers, which probably explains why it was missed by genetic mapping studies.     

Survey, analysis and genetic organization of genes encoding eukaryotic-like signaling proteins on a cyanobacterial genome.

Bacteria usually use two-component systems for signal transduction, while eukaryotic organisms employ Ser/Thr and Tyr kinases and phosphatases for the same purpose. Many prokaryotes turn out to harbor Ser/Thr and Tyr kinases, Ser/Thr and Tyr phosphatases, and their accessory components as well. The sequence determination of the genome of the cyanobacterium Synechocystis sp. strain PCC 6803 offers the possibility to survey the extent of such molecules in a prokaryotic organism. This cyanobacterium possesses seven Ser/Thr kinases, seven Ser/Thr and Tyr phosphatases, one protein kinase interacting protein, one protein kinase regulatory subunit and several WD40-repeat-containing proteins. The majority of the protein phosphatases presented in this study were previously reported as hypothetical proteins. We analyze here the structure and genetic organization of these ORFs in the hope of providing a guidance for their functional analysis. Unlike their eukaryotic counterparts, many of these genes are clustered on the chromosome, and this genetic organization offers the opportunity to study their possible interaction. In several cases, genes of two-component transducers are found within the same cluster as those encoding a Ser/Thr kinase or a Ser/Thr phosphatase; the implication for signal transduction mechanism will be discussed.     

Complete nucleotide sequence and genome organization of a Drosophila transposable genetic element, 297

The complete nucleotide sequence of 297, a Drosophila copia-like transposable element, was determined and compared with those of other similar Drosophila elements and mammalian retrovirus proviruses. It was found that 297 contains three long open reading frames, comparable in sizes and locations with gag, pol, and env genes in the proviruses of replication-competent retroviruses in vertebrates. The first and second open reading frames of 297 exhibit sequence homologies to gag and pol, respectively, of Moloney murine leukaemia virus. In particular, as with 17.6, another Drosophila copia-like element, the second open reading frame of 297 was shown to be very similar in its entire organization to the retroviral pol gene and to consist of three enzymatic domains. By contrast, no appreciable homology was found between the third open reading frame of 297 and the retroviral env gene. It is also suggested that 297 and 17.6 are a peculiar pair of copia-like elements recently diverged from a common progenitor.     

The Aedes aegypti genome: complexity and organization.

We describe the use of DNA reassociation kinetics to determine the total genome size and complexity together with the individual complexity and copy number of the single copy, middle repetitive and highly repeated DNA fractions of cell line and larval DNA from the mosquito, Aedes aegypti. The genome of Ae. aegypti is both large and complex, being one third the size of the human genome, and exhibits a short period interspersed repeat pattern. The implications of patterns of sequence arrangement and genome complexities for experiments aimed at isolating specific classes of DNA sequences, such as mobile genetic elements, are discussed.     

An integrated physical and genetic map of the rice genome

Rice was chosen as a model organism for genome sequencing because of its economic importance, small genome size, and syntenic relationship with other cereal species. We have constructed a bacterial artificial chromosome fingerprint–based physical map of the rice genome to facilitate the whole-genome sequencing of rice. Most of the rice genome (~90.6%) was anchored genetically by overgo hybridization, DNA gel blot hybridization, and in silico anchoring. Genome sequencing data also were integrated into the rice physical map. Comparison of the genetic and physical maps reveals that recombination is suppressed severely in centromeric regions as well as on the short arms of chromosomes 4 and 10. This integrated high-resolution physical map of the rice genome will greatly facilitate whole-genome sequencing by helping to identify a minimum tiling path of clones to sequence. Furthermore, the physical map will aid map-based cloning of agronomically important genes and will provide an important tool for the comparative analysis of grass genomes.     

The human genome structure and organization

Genetic information of human is encoded in two genomes: nuclear and mitochondrial. Both of them reflect molecular evolution of human starting from the beginning of life (about 4.5 billion years ago) until the origin of Homo sapiens species about 100,000 years ago. From this reason human genome contains some features that are common for different groups of organisms and some features that are unique for Homo sapiens. 3.2 x 10(9) base pairs of human nuclear genome are packed into 23 chromosomes of different size. The smallest chromosome - 21st contains 5 x 10(7) base pairs while the biggest one -1st contains 2.63 x 10(8) base pairs. Despite the fact that the nucleotide sequence of all chromosomes is established, the organisation of nuclear genome put still questions: for example: the exact number of genes encoded by the human genome is still unknown giving estimations from 30 to 150 thousand genes. Coding sequences represent a few percent of human nuclear genome. The majority of the genome is represented by repetitiVe sequences (about 50%) and noncoding unique sequences. This part of the genome is frequently wrongly called "junk DNA". The distribution of genes on chromosomes is irregular, DNA fragments containing low percentage of GC pairs code lower number of genes than the fragments of high percentage of GC pairs.     

The nuclear lamina. Both a structural framework and a platform for genome organization

The inner face of the nuclear envelope of metazoan cells is covered by a thin lamina consisting of a one-layered network of intermediate filaments interconnecting with a complex set of transmembrane proteins and chromatin associating factors. The constituent proteins, the lamins, have recently gained tremendous recognition, because mutations in the lamin A gene, LMNA, are the cause of a complex group of at least 10 different diseases in human, including the Hutchinson-Gilford progeria syndrome. The analysis of these disease entities has made it clear that besides cytoskeletal functions, the lamina has an important role in the "behaviour" of the genome and is, probably as a consequence of this function, intimately involved in cell fate decisions. Furthermore, these functions are related to the involvement of lamins in organizing the position and functional state of interphase chromosomes as well as to the occurrence of lamins and lamina-associated proteins within the nucleoplasm. However, the structural features of these lamins and the nature of the factors that assist them in genome organization present an exciting challenge to modern biochemistry and cell biology.     

Mitochondrial genetics in perspective: the derivation of a genetic and physical map of the yeast mitochondrial genome.

The attainment of the map of functions coded in the yeast mitochondrial genome represents the end of an era of development in mitochondrial genetics. Following the earliest genetic studies, where first the respiration-deficient petite mutants, then subsequently the other types of mitochondrial mutants, were characterized, it was realized that a genetic approach to the questions of mitochondrial biogenesis and the genetic function of mtDNA would yield much useful information. A period of intensive investigation into the behavior of mitochondrial genes in genetic crosses followed, and it was concluded that the purely genetic techniques of transmissional and recombinational analysis could not yield a map of the genetic loci, although basic rules for mitochondrial genetic manipulation were established. The concurrent studies of the nature of the deletions in petite mtDNA led to the recognition that an analysis of the behavior of genetic loci in petite mutants would provide the method for genetically mapping the positions of loci in mtDNA where conventional genetic crosses between grande strains had failed. This thesis was first confirmed by our studies of the frequencies of coretention and loss of individual loci in large populations of petite isolates, which produced the first circular genetic map of drug resistance loci on mtDNA. Subsequent to this genetic mapping phase, we established a general procedure for determining the physical map position of any mitochondrial genetic locus or mtDNA sequence by introducing the use of a molecular library of petite mutants carrying physically and genetically defined segments of mtDNA. These petites can be tested for the retention or loss of genetic loci or particular nucleotide sequences. This general solution to the mapping problem and the physical map of the Saccharomyces cerevisiae mitochondrial genome obtained, which has been confirmed by studies using restriction enzymes, has provided the field with a molecular point of reference for the many current genetic and biochemical investigations into the structure and function of mtDNA in yeast.     

PFGE analysis of the rice genome: estimation of fragment sizes,organization of repetitive sequences and relationships between genetic and physical distances

Pulsed-field gel electrophoresis (PFGE) has been applied to analyze the rice nuclear genome. Probing 56 RFLP probes selected from the 12 rice chromosomes to PFGE blots of nine rare-cutting restriction enzymes revealed that there are relatively high numbers of rare-cutting restriction sites in the rice genome. The average sizes of restriction fragments detected by single-copy probes are smaller than 200 kb for all of the rare-cutting restriction enzymes examined. Sizes of fragments detected by repetitive probes are variable, depending on the probes analyzed. By using PFGE, a tandemly repeated sequence, Os48, was found to be tightly linked to telomeric tandem repeats but not physically linked to r5s genes with which sequence homology had been observed. Relationships between genetic and physical distances have been established for three different chromosomal segments. In these regions 1 cm corresponds to ca. 260 kb on average. Analysis of a cluster of RFLP markers on chromosome 3 revealed that genetically clustered RFLP markers are also physically closely linked, suggesting that clustering of genetic markers may result in part from uneven distribution of single-copy sequences.     

The organization and inheritance of the mitochondrial genome

Mitochondrial DNA (mtDNA) encodes essential components of the cellular energy-producing apparatus, and lesions in mtDNA and mitochondrial dysfunction contribute to numerous human diseases. Understanding mtDNA organization and inheritance is therefore an important goal. Recent studies have revealed that mitochondria use diverse metabolic enzymes to organize and protect mtDNA, drive the segregation of the organellar genome, and couple the inheritance of mtDNA with cellular metabolism. In addition, components of a membrane-associated mtDNA segregation apparatus that might link mtDNA transmission to mitochondrial movements are beginning to be identified. These findings provide new insights into the mechanisms of mtDNA maintenance and inheritance.     

An improved physical and genetic map of the genome of alkaliphilic Bacillus sp. C-125

Among alkaliphilic bacteria reported so far, Bacillus sp. C-125 is the strain most thoroughly characterized physiologically, biochemically, and genetically. A physical map of the chromosome of this strain was constructed to facilitate further genome analysis, and the genome size was revised from 3.7 to 4.25 Mb. Complete digestion of the chromosomal DNA with two rare cut restriction endonucleases, AscI and Sse8387I, each yielded 20 fragments ranging in size from 20 to 600 kb. Seventeen linking clones were isolated in each instance to join the adjacent AscI or Sse8387I fragments in the chromosomal map. All AscI linking clones isolated were sequenced and analyzed by comparison with the BSORF database to map the genes in the chromosome of strain C-125. Several ORFs showing significant similarities to those of B. subtilis in the AscI linking clones were positioned on the physical map. The oriC region of the C-125 chromosome was identified by southern blot analysis with a DNA probe containing the gyrB region. Received: May 6, 1998 / Accepted: May 26, 1998     

A physical and genetic map of the Mycoplasma hyopneumoniae strain J genome

A macrorestriction map of the genome of Mycoplasma hyopneumoniae strain J, the type strain of the causative agent of enzootic pneumonia in pigs, was constructed using pulsed-field gel electrophoresis (PFGE) and DNA hybridization. The size of the genome as determined by PFGE was approximately 1070 kb. Assembly of the M. hyopneumoniae genomic map was facilitated and complimented by the simultaneous construction of an ordered cosmid library. Five contigs of overlapping cosmids were assembled, which together represent coverage of approximately 728 kb. Forty-two genetic markers (including three types of repeated elements) were placed on the M. hyopneumoniae map. Closer examination of an ApaI restriction fragment contained entirely within a single cosmid insert suggests that the genome size may be overestimated by PFGE.