Spline methods for the comparison of physical and genetic maps.

The first genetic maps were constructed by linkage analysis. Physical mapping techniques, such as radiation hybrids and complete sequencing, produce a different picture. For the purposes of population genetics, clinical genetics, and genetic epidemiology, it is important to harmonize and amalgamate existing genetic and physical maps. Among other things, comparisons of the two kinds of maps promotes better understanding of the wide variation in local recombination rates per unit physical length of DNA. The current paper presents methods for estimating recombination intensity as a function of physical distance along a chromosome. Genetic map distance is the integral of intensity. We derive fast reliable estimation algorithms based on a Poisson process model, penalized likelihoods, and cubic spline interpolation. Our methods provide a rigorous and statistically sound foundation for comparing physical and genetic maps. To illustrate the possibilities, we apply the methods to published recombination data on CEPH families and the complete sequences of chromosomes 21 and 22. Our results are in good agreement with previous studies and the biological data.     

The frame of non-canonical theory of heredity: from genes to epigenes

Particular theory of heredity that exceeds the limits of mendelian genetics is suggested. The model based on five sufficiently obvious assumptions (accepted as axioms) As consequence of these axioms the strict statements concerningfunctional heredity memory were formulated in mathematical terms. Molecular-genetic realization of the memory cells appears as new class of heredity units--epigenes. In the epigenes part f hereditary information is contained, encoded and transmitted beyond the primary structure of DNA molecules of genome. Epigenes capable to conserve sequences of genes functional states in the course of ontogenesis and provide transmission of information contained in this states throw consequent generations. It was shown that epigenes differ from genes at least by encoding method of heredity information. There are three functional-equivalent classes of really existing epigenes mechanisms: dynamic, modificational and transpositional; and there is one hypothetical class--invertional. It was shown that a lot of experimental data concerning epigenetic mechanism of heredity is in accord with theoretical conclusions concerning epigenes existence. Moreover, we constructed an artificial epigenes by genetic engineering methods. The existence of epigenes means that obtaining complete genome sequence, its physical and genetic maps, as well as distinguishing the rules of genes function encoding by its primary structure do not provide complete decoding of hereditary information. The role of epigenes in ontogenesis and phylogenesis was examined. It was shown that even elementary epigenetic systems could determine key ontogenesis events. Epigenetic system could serve as the basis of non-darwinian evolutionary strategies by means of "memorization of rather unsuccessfully steps of evolution" and conservation of alternative variants of ontogenesis. Teleonomic hypothesis on functional heredity memory was formulated. This theory provides explanation of phenomena of acquired features inheritance and molecular mechanisms of stress-induced evolution.     

Map error reduction: using genetic and sequence-based physical maps to order closely linked markers

The Marshfield comprehensive genetic maps are frequently used for linkage and association studies, however, for some regions of these maps the marker order has low level of likelihood ratio support. In order to investigate the level of statistical support and the accuracy of the genetic maps compared to sequence-based physical maps, two approximately 30 cM autosomal regions were selected. The first region was selected from chromosome 3 and consisted predominately of draft sequence. The second region was selected from chromosome 21 and consisted of finished sequence data. The physical order of these markers was based upon their position on Celera (CEL) and Human Genome Project-Santa Cruz (HGP-sc) sequence-based physical maps. The chromosome 3 and 21 regions contained 100 and 61 markers, respectively, on the Marshfield genetic map. The genetic and physical map order was consistent for 88.9 and 89.2% of the markers in the region on chromosome 3 and 21, respectively. Using a novel scoring criterion to assess inconsistent marker order between genetic and physical maps, it was determined that the physical order was likely the correct order for 3.3 and 7.1% of the markers in the chromosome 3 and 21 regions, respectively. To increase the accuracy of the order of markers selected for fine mapping a method is presented which combines information from genetic and sequence-based physical maps.     

New techniques for physical mapping of the human genome.

We describe improvements in techniques and strategies used for making maps of the human genome. The methods currently used are changing and evolving rapidly. Today's techniques can produce ordered arrays of DNA fragments and overlapping sets of DNA clones covering extensive genomic regions, but they are relatively slow and tedious. Methods under development will speed the process considerably. New developments include a range of applications of the polymerase chain reaction, enhanced procedures for high resolution in situ hybridization, and improved methods for generating, manipulating, and cloning large DNA fragments. More detailed genetic and physical maps will be useful for finding genes, including those associated with human diseases, long before the complete DNA sequence of the human genome is available.     

DNA shape, genetic codes, and evolution

Although the three-letter genetic code that maps nucleotide sequence to protein sequence is well known, there must exist other codes that are embedded in the human genome. Recent work points to sequence-dependent variation in DNA shape as one mechanism by which regulatory and other information could be encoded in DNA. Recent advances include the discovery of shape-dependent recognition of DNA that depends on minor groove width and electrostatics, the existence of overlapping codes in protein-coding regions of the genome, and evolutionary selection for compensatory changes in nucleotide composition that facilitate nucleosome occupancy. It is becoming clear that DNA shape is important to biological function, and therefore will be subject to evolutionary constraint.     

Alignment of Escherichia coli K12 DNA sequences to a genomic restriction map.

We use the extensive published information describing the genome of Escherichia coli and new restriction map alignment software to align DNA sequence, genetic, and physical maps. Restriction map alignment software is used which considers restriction maps as strings analogous to DNA or protein sequences except that two values, enzyme name and DNA base address, are associated with each position on the string. The resulting alignments reveal a nearly linear relationship between the physical and genetic maps of the E. coli chromosome. Physical map comparisons with the 1976, 1980, and 1983 genetic maps demonstrate a better fit with the more recent maps. The results of these alignments are genomic kilobase coordinates, orientation and rank of the alignment that best fits the genetic data. A statistical measure based on extreme value distribution is applied to the alignments. Additional computer analyses allow us to estimate the accuracy of the published E. coli genomic restriction map, simulate rearrangements of the bacterial chromosome, and search for repetitive DNA. The procedures we used are general enough to be applicable to other genome mapping projects.     

Funnel-like organization in sequence space determines the distributions of protein stability and folding rate preferred by evolution

To understand the physical and evolutionary determinants of protein folding, we map out the complete organization of thermodynamic and kinetic properties for protein sequences that share the same fold. The exhaustive nature of our study necessitates using simplified models of protein folding. We obtain a stability map and a folding rate map in sequence space. Comparison of the two maps reveals a common organizational principle: optimality decreases more or less uniformly with distance from the optimal sequence in the sequence space. This gives a funnel-shaped optimality surface. Evolutionary dynamics of a sequence population on these two maps reveal how the simple organization of sequence space affects the distributions of stability and folding rate preferred by evolution.     

DNA fiber mapping techniques for the assembly of high-resolution physical maps.

High-resolution physical maps are indispensable for directed sequencing projects or the finishing stages of shotgun sequencing projects. These maps are also critical for the positional cloning of disease genes and genetic elements that regulate gene expression. Typically, physical maps are based on ordered sets of large insert DNA clones from cosmid, P1/PAC/BAC, or yeast artificial chromosome (YAC) libraries. Recent technical developments provide detailed information about overlaps or gaps between clones and precisely locate the position of sequence tagged sites or expressed sequences, and thus support efforts to determine the complete sequence of the human genome and model organisms. Assembly of physical maps is greatly facilitated by hybridization of non-isotopically labeled DNA probes onto DNA molecules that were released from interphase cell nuclei or recombinant DNA clones, stretched to some extent and then immobilized on a solid support. The bound DNA, collectively called "DNA fibers," may consist of single DNA molecules in some experiments or bundles of chromatin fibers in others. Once released from the interphase nuclei, the DNA fibers become more accessible to probes and detection reagents. Hybridization efficiency is therefore increased, allowing the detection of DNA targets as small as a few hundred base pairs. This review summarizes different approaches to DNA fiber mapping and discusses the detection sensitivity and mapping accuracy as well as recent achievements in mapping expressed sequence tags and DNA replication sites.     

Integration of the Aedes aegypti mosquito genetic linkage and physical maps

Two approaches were used to correlate the Aedes aegypti genetic linkage map to the physical map. STS markers were developed for previously mapped RFLP-based genetic markers so that large genomic clones from cosmid libraries could be found and placed to the metaphase chromosome physical maps using standard FISH methods. Eight cosmids were identified that contained eight RFLP marker sequences, and these cosmids were located on the metaphase chromosomes. Twenty-one cDNAs were mapped directly to metaphase chromosomes using a FISH amplification procedure. The chromosome numbering schemes of the genetic linkage and physical maps corresponded directly and the orientations of the genetic linkage maps for chromosomes 2 and 3 were inverted relative to the physical maps. While the chromosome 2 linkage map represented essentially 100% of chromosome 2, approximately 65% of the chromosome 1 linkage map mapped to only 36% of the short p-arm and 83% of the chromosome 3 physical map contained the complete genetic linkage map. Since the genetic linkage map is a RFLP cDNA-based map, these data also provide a minimal estimate for the size of the euchromatic regions. The implications of these findings on positional cloning in A. aegypti are discussed.     

Comparative DNA sequence analysis of mapped wheat ESTs reveals the complexity of genome relationships between rice and wheat

The use of DNA sequence-based comparative genomics for evolutionary studies and for transferring information from model species to related large-genome species has revolutionized molecular genetics and breeding strategies for improving those crops. Comparative sequence analysis methods can be used to cross-reference genes between species maps, enhance the resolution of comparative maps, study patterns of gene evolution, identify conserved regions of the genomes, and facilitate interspecies gene cloning. In this study, 5,780 Triticeae ESTs that have been physically mapped using wheat (Triticum aestivum L.) deletion lines and segregating populations were compared using NCBI BLASTN to the first draft of the public rice (Oryza sativa L.) genome sequence data from 3,280 ordered BAC/PAC clones. A rice genome view of the homoeologous wheat genome locations based on sequence analysis shows general similarity to the previously published comparative maps based on Southern analysis of RFLP. For most rice chromosomes there is a preponderance of wheat genes from one or two wheat chromosomes. The physical locations of non-conserved regions were not consistent across rice chromosomes. Some wheat ESTs with multiple wheat genome locations are associated with the non-conserved regions of similarity between rice and wheat. The inverse view, showing the relationship between the wheat deletion map and rice genomic sequence, revealed the breakdown of gene content and order at the resolution conferred by the physical chromosome deletions in the wheat genome. An average of 35% of the putative single copy genes that were mapped to the most conserved bins matched rice chromosomes other than the one that was most similar. This suggests that there has been an abundance of rearrangements, insertions, deletions, and duplications eroding the wheat-rice genome relationship that may complicate the use of rice as a model for cross-species transfer of information in non-conserved regions.     

On the origin and evolution of the genetic code

Two ideas have essentially been used to explain the origin of the genetic code: Crick's frozen accident and Woese's amino acid-codon specific chemical interaction. Whatever the origin and codon-amino acid correlation, it is difficult to imagine the sudden appearance of the genetic code in its present form of 64 codons coding for 20 amino acids without appealing to some evolutionary process. On the contrary, it is more reasonable to assume that it evolved from a much simpler initial state in which a few triplets were coding for each of a small number of amino acids. Analysis of genetic code through information theory and the metabolism of pyrimidine biosynthesis provide evidence that suggests that the genetic code could have begun in an RNA world with the two letters A and U grouped in eight triplets coding for seven amino acids and one stop signal. This code could have progressively evolved by making gradual use of letters G and C to end with 64 triplets coding for 20 amino acids and three stop signals. According to proposed evidence, DNA could have appeared after the four-letter structure was already achieved. In the newborn DNA world, T substituted U to get higher physicochemical and genetic stability.     

Identification and High-Density Mapping of Gene-Rich Regions in Chromosome Group 5 of Wheat

The distribution of genes and recombination in the wheat genome was studied by comparing physical maps with the genetic linkage maps. The physical maps were generated by mapping 80 DNA and two phenotypic markers on an array of 65 deletion lines for homoeologous group 5 chromosomes. The genetic maps were constructed for chromosome 5B in wheat and 5D in Triticum tauschii. No marker mapped in the proximal 20% chromosome region surrounding the centromere. More than 60% of the long arm markers were present in three major clusters that physically encompassed <18% of the arm. Because 48% of the markers were cDNA clones and the distributions of the cDNA and genomic clones were similar, the marker distribution may represent the distribution of genes. The gene clusters were identified and allocated to very small chromosome regions because of a higher number of deletions in their surrounding regions. The recombination was suppressed in the centromeric regions and mainly occurred in the gene-rich regions. The bp/cM estimates varied from 118 kb for gene-rich regions to 22 Mb for gene-poor regions. The wheat genes present in these clusters are, therefore, amenable to molecular manipulations parallel to the plants with smaller genomes like rice.     

Heterology of mitochondrial DNA from mammals detected by electron microscopic heteroduplex analyses.

Heteroduplex analysis of mitochondrial DNA (mtDNA) from evolutionary closely related mammals (rat vs. mouse, man vs. monkey) are analyzed and compared to heteroduplex analysis of mt-DNA from more distantly related mammals (rat vs. man, rat vs. monkey, mouse vs. man, mouse vs. monkey and man vs. cow). Each analysis is transformed into a heteroduplex map and all maps are aligned to restriction enzyme maps and to genetic maps and where possible compared with the known sequence. We show that early evolutionary changes are seen mainly in URF2, URFA6L, URF6 and the D-loop region. The regions of rRNA, URF1, COI and COIII are generally very conserved regions but areas with some evolutionary activity can be localized. Heteroduplex analysis between distantly related species show much more heterology than do closely related species and the heteroduplex maps between all the distantly related species show a common pattern of heterology. Comparisons between the DNA sequence of mtDNA from man, cow and mouse and the equivalent heteroduplex maps show that base pair homologies higher than 73% are displayed as homologous regions. In the heteroduplex analysis of mtDNA's from more closely related species very few heterologies are displayed at 50% formamide but an increase in formamide concentration to 65-70% demonstrate also in these instances general heterologous regions.     

Comparative maps: adding pieces to the mammalian jigsaw puzzle

Comparative maps display the chromosomal location of homologous genes in different species and highlight genetic segments that are conserved in evolution. These maps are used to study chromosomal changes that occurred during the divergence of mammalian lineages, to identify candidates for hereditary disease genes, and to facilitate mapping in other species. Recently, physical mapping in regions of known conserved linkage has revealed previously undetected chromosomal changes that may provide clues to understanding chromosomal structure and function and evolutionary processes. The availability of these data in electronically accessible formats is critical to the growth and analysis of comparative maps.     

The distribution of RFLP markers on chromosome 2(2H) of barley in relation to the physical and genetic location of 5S rDNA

The 5S rDNA locus on the long arm of barley chromosome 2(2H) was genetically mapped in two crosses in relation to 30 other RFLP loci. Comparison of the genetic maps with the previously published physical position of the 5S rDNA, determined by in-situ hybridization, showed that there was a marked discrepancy between physical and genetic distance in both crosses, with recombination being less frequent in the proximal part of the arm. Pooled information from the present study and other published genetic maps showed that at least 26 of the 44 (59%) RFLPs that have been mapped on 2(2H)L lie distal to the 5S rDNA locus even though this region is only 27% of the physical length of the arm. The distribution of RFLP markers is significantly different from expected (P < 0.01), implying that the low-copy sequences used for RFLP analysis occur more frequently in distal regions of the arm and, or, that sequences in distal regions are more polymorphic.     

Genetic linkage analysis and homology relationships of genes located on human chromosome 11q.

We have used DNA polymorphisms detected by probes for 11q to order 16 genes and to determine the genetic distances between them. Our map includes the genes for CD20, tyrosinase, progesterone receptor, stromelysin, collagenase, N-CAM, dopamine-D2 receptor, apolipoproteins AI-CIII-AIV, CD3-epsilon, -delta, and -gamma, porphobilinogen deaminase, thy-1, and ets-1. These genes have previously been sequenced as well as placed on the 11q cytogenetic map, which now makes them anchor points between the cytogenetic, genetic, and physical maps of this region. The ordering and distances between these genes are of immediate use in testing hypotheses of candidate genes for human genetic diseases associated with chromosome 11q. A comparison between our genetic map and similar maps from other species defines regions of homologous synteny that may be useful in mapping human genetic disease genes localized to the 11q region. Analysis of such homology provides additional bases for speculation of the evolutionary histories of gene families in this region.     

Physical mapping of beta-converting and gamma-nonconverting corynebacteriophage genomes

Deoxyribonucleic acids (DNAs) from wild-type and mutant strains of beta-converting and gamma-nonconverting corynebacteriophages were isolated and physically characterized. The data obtained from DNA heteroduplexes, restriction enzyme banding profiles, and restriction maps reinforce the conclusion that beta and gamma phages are very closely related. The major physical differences seen in the DNA heteroduplexes are a small substitution bubble and one or two insertions which are present on the gamma phage genome. The insertions account for the differences in the genome sizes of beta and gamma phages, and with the substitution they are responsible for most of the differences in the restriction endonuclease profiles and maps of the corynebactriophage genomes, two special sites and the DNA fragments carrying them were identified. These were the cohesive (cos) sites and the specific attachment (attP) site of the vegetative phage genome. The behavior of these sites indicated that the transition of phage DNA from the vegetative to the prophage state involves the circularization of vegetative DNA through the cos sites and its integration into the bacterial chromosome via the attP site. The mechanism of corynebacteriophage integration was similar to that employed by Escherichia coli phage gamma. From the data assembled the physical and genetic maps of beta and gamma phage were oriented with respect to one another. The extensive similarity in their maps provides additional confirmation of a close evolutionary relationship.     

Stability distribution in the phage lambda-DNA double helix: a correlation between physical and genetic structure

Statistical analyses on the positional correlation of physical-stability and base-sequence distribution maps with genetic map are made for the whole DNA (48502 bases) of lambda-phage. The susceptibility to a double-helix unfolding perturbation and the fraction of the transient opening of a particular region of the double helix are adopted to define this physical stability. The principal features obtained are: A) The DNA double strand of protein coding regions is found to have homostabilizing propensity around a defined stability which is characteristic to each individual gene. B) The stability of the double helix in non-protein coding region fluctuates, on average over the whole region, more than that in protein coding region. C) Boundary regions of protein coding and non-protein coding regions are regions of high stability-fluctuation. Stability especially fluctuates at the protein-coding-region side of the boundary. Contrary to the quiet feature of the interior part of protein coding region rather noisy part exists at its edge. D) One frequently opening region coincides with the attaching site for the site specific recombination between phage and bacterial DNA. There are two possible ways to explain the noisy feature in the stability distribution in non-protein coding regions: 1) The region has been used as the locus of recombination as evolution took place. Thus DNAs which were homostabilized around a different value characteristic to each individual DNA, have been joined there many times, so that the noise has accumulated as a remnant of evolutional history; and/or 2) the base-composition homogenizing or double-helix homostabilizing mechanism does not work in unneeded region such as non-protein coding region or introns. Since corresponding characteristics have been found in our previous analyses on other viral and globin-gene DNAs, the rules mentioned above may be comprehensively extended to other DNAs.     

Heterogeneity in Rates of Recombination across the Mouse Genome

If loci are randomly distributed on a physical map, the density of markers on a genetic map will be inversely proportional to recombination rate. First proposed by MARY LYON, we have used this idea to estimate recombination rates from the Drosophila melanogaster linkage map. These results were compared with results of two other studies that estimated regional recombination rates in D. melanogaster using both physical and genetic maps. The three methods were largely concordant in identifying large-scale genomic patterns of recombination. The marker density method was then applied to the Mus musculus microsatellite linkage map. The distribution of microsatellites provided evidence for heterogeneity in recombination rates. Centromeric regions for several mouse chromosomes had significantly greater numbers of markers than expected, suggesting that recombination rates were lower in these regions. In contrast, most telomeric regions contained significantly fewer markers than expected. This indicates that recombination rates are elevated at the telomeres of many mouse chromosomes and is consistent with a comparison of the genetic and cytogenetic maps in these regions. The density of markers on a genetic map may provide a generally useful way to estimate regional recombination rates in species for which genetic, but not physical, maps are available.