Staphylococcus aureus has clustered tRNA genes.

The polymerase chain reaction (PCR) was used to detect large tRNA gene clusters in Bacillus subtilis, Bacillus badius, Bacillus megaterium, Lactobacillus brevis, Lactobacillus casei, and Staphylococcus aureus. The primers were based on conserved sequences of known gram-positive bacterial tRNA(Arg) and tRNA(Phe) genes. This PCR procedure detected an unusually large tRNA gene cluster in S. aureus. PCR-generated probes were used to identify a 4.5-kb EcoRI fragment that contained 27 tRNA genes immediately 3' to an rRNA operon. Some of these 27 tRNA genes are very similar, but only 1 is exactly repeated in the cluster. The 5' end of this cluster has a gene order similar to that found in the 9- and 21-tRNA gene clusters of B. subtilis. The 3' end of this S. aureus cluster exhibits more similarity to the 16-tRNA gene cluster of B. subtilis. The 24th, 25th, and 26th tRNA genes of this S. aureus tRNA gene cluster code for three similar, unusual Gly-tRNAs that may be used in the synthesis of the peptidoglycan in the cell wall but not in protein synthesis. Southern analysis of restriction digests of S. aureus DNA indicate that there are five to six rRNA operons in this bacterium's genome and that most or all may have large tRNA gene clusters at the 3' end.     

The diversity of connexin genes encoding gap junctional proteins.

The multigene family of connexins is larger than previously anticipated. Ten different connexin homologous sequences have been characterized in the mouse genome, five of which are probably the mouse analogues of the known rat connexins26, -31, -32, -43, and -46. Since the additional 5 sequences have been isolated as cDNAs or hybridize specifically to distinct mRNA species, they most likely represent functional connexin genes. Since seven of the genomic connexin sequences have been shown to contain no intron in the coding sequence, this may apply to all mammalian connexin genes. Some of the structural features based on amino acid sequences deduced from cDNA or genomic sequences and the RNA expression pattern of the new connexins are compared with previously described connexins. The structural diversity of the connexin genes suggests that they fulfill different functions coordinated with, and perhaps required for, different programs of cellular differentiation.     

The statistical analysis of density dependence.

The distrubution of the reciprocal of von Neumann's ratio is considered under the random walk model, x(t+1) rwis;d xt + et, and it is shown how this quantity can be used to test for density dependence in animal populations. Another test is described which is robust under superimposed errors of measurement. The methods are used to analyze data on Canadian fur-bearing mammals.     

The role of chromatin structure in regulating the expression of clustered genes

Much of what we know about the chromatin-based mechanisms that regulate gene expression in mammals has come from the study of what are, paradoxically, atypical genes. These are clusters of structurally and/or functionally related genes that are coordinately regulated during development, or between different cell types. Can unravelling the mechanisms of gene regulation at these gene clusters help us to understand how other genes are controlled? Moreover, can it explain why there is clustering of apparently unrelated genes in mammalian genomes?     

The structure of the human histone genes: clustered but not tandemly repeated

We have isolated five clones containing human histone genes from a human genomic DNA library, using a cloned X. laevis histone H4 cDNA probe (pX1ch4). Each genomic clone has been mapped and the locations of the histone genes have been determined by blot hybridization and hybridization-selection of human histone mRNA. In contrast to the organization of previously characterized histone genes, the human histone genes are clustered in the genome but are not arranged into recognizable repeating units. The extreme lack of organization of the human histone genes may reflect the diminished requirement for rapid synthesis of large quantities of histone proteins during mammalian early development.     

Free energy gap and statistical thermodynamic fidelity of DNA codes.

DNA nanotechnology often requires collections of oligonucleotides called "DNA free energy gap codes" that do not produce erroneous crosshybridizations in a competitive muliplexing environment. This paper addresses the question of how to design these codes to accomplish a desired amount of work within an acceptable error rate. Using a statistical thermodynamic and probabilistic model of DNA code fidelity and mathematical random coding theory methods, theoretical lower bounds on the size of DNA codes are given. More importantly, DNA code design parameters (e.g., strand number, strand length and sequence composition) needed to achieve experimental goals are identified.     

A simple method for the analysis of clustered binary data.

A simple method for comparing independent groups of clustered binary data with group-specific covariates is proposed. It is based on the concepts of design effect and effective sample size widely used in sample surveys, and assumes no specific models for the intracluster correlations. It can be implemented using any standard computer program for the analysis of independent binary data after a small amount of preprocessing. The method is applied to a variety of problems involving clustered binary data: testing homogeneity of proportions, estimating dose-response models and testing for trend in proportions, and performing the Mantel-Haenszel chi-squared test for independence in a series of 2 x 2 tables and estimating the common odds ratio and its variance. Illustrative applications of the method are also presented.     

Comparative genomics and diversifying selection of the clustered vertebrate protocadherin genes

To explain the mechanism for specifying diverse neuronal connections in the brain, Sperry proposed that individual cells carry chemoaffinity tags on their surfaces. The enormous complexity of these connections requires a tremendous diversity of cell-surface proteins. A large number of neural transmembrane protocadherin (Pcdh) proteins is encoded by three closely linked human and mouse gene clusters (alpha, beta, and gamma). To gain insight into Pcdh evolution, I performed comprehensive comparative cDNA and genomic DNA analyses for the three clusters in the chimpanzee, rat, and zebrafish genomes. I found that there are species-specific duplications in vertebrate Pcdh genes and that additional diversity is generated through alternative splicing within the zebrafish "variable" and "constant" regions. Moreover, different codons (sites) in the mammalian Pcdh ectodomains (ECs) are under diversifying selection, with some under diversity-enhancing positive Darwinian selection and others, including calcium-binding sites, under strong purifying selection. Interestingly, almost all positively selected codon positions are located on the surface of ECs 2 and 3. These diversified residues likely play an important role in combinatorial interactions of Pcdh proteins, which could provide the staggering diversity required for neuronal connections in the brain. These results also suggest that adaptive selection is an additional evolutionary factor for increasing Pcdh diversity.     

Evolution of EF-hand calcium-modulated proteins. V. The genes encoding EF-hand proteins are not clustered in mammalian genomes

Summary The chromosomal assignments of genes belonging to the EF-hand family which have a common origin are compiled in this article. So far data are available from 27 human gene loci belonging to 6 subfamilies and 8 murine loci belonging to 4 subfamilies. Chromosomal localization has been obtained by somatic-cell hybrid analysis using the Southern blot technique or PCR amplification, metaphase spread in situ hybridization, or isolation of the particular genes from chromosome-specific libraries. Except for genes of the S-100 alpha proteins which are grouped on human chromosome 1q12-25 and mouse chromosome 3, no linkage has been found for genes encoding EF-hand proteins, indicating absence of selective pressure for maintaining chromosomal clustering. Six of these genes map to known syntenic groups conserved in the human and mouse genomes. This suggests that chromosomal translocations occurred before divergence of these species. The possible significance of chromosomal positioning with respect to nearby located known genes and genetic disease loci is discussed.     

The phylogeny of human globin genes investigated by the maximum parsimony method

Gene phylogenetic trees were constructed by the maximum parsimony method for various sets of ninety six globin chain amino acid sequences spanning plant and animal kingdoms. The method, executed by several computer programs, constructed ancestor and descendant globin messengers on tree topologies which required the least number of nucleotide replacements to account for the evolution of the globins. The human myoglobin-hemoglobin divergence was traced to a gene duplication which occurred either in the first vertebrates or earlier yet in the common ancestor of chordates and annelids, the alpha-beta divergence to a gene duplication in the common ancestor of teleosts and tetrapods, the gamma divergence from typical beta chains to a gene duplication in basal therian mammals, and the delta separation from beta to a duplication in the basal catarrhine primates. Evidence was provided by the globin phylogenies for the hominoid affinities of the gibbon and the close phyletic relationship of the African apes to man. Over the period of teleos-tetrapod divergence the globin messengers evolved at an average rate of 18.5 nucleotide replacements per 100 codons per 10⁸ years, a faster rate than most previous estimates. Very fast and very slow rates were encountered in different globin lineages and at different stages of descent, reducing the effectiveness of globins as molecular clocks. Rates increased with gene duplication and decreased after selection discovered useful specializations in the products of genes which had previously been freer to accept mutations. The early eutherian radiation was characterized by rapid rates of globin evolution, but the later hominoid radiation by extremely slow rates. This pattern was related to more complicated grades of internal organization evolving in human ancestors. The types of nucleotide replacements in the globin messengers over the long course of globin evolution did not seem indicative of any special mutational mechanisms.     

The statistical analysis of the differential blood count

The differential blood count obtained by unbiased sampling mathematically follows a multinomial distribution. The variation between individuals can be formalized by a mixing distribution of the parameters of the multinomial distribution. By DIRICHLET-distributions used as mixing distributions the main phenomena of the observed data can be described, and they are useful in estimating and testing treatment effects. If the correlation between the different types of leucocytes is taken into account in an appropriate manner, univariate test procedures can be applied also.     

Identification of novel universal housekeeping genes by statistical analysis of microarray data

Housekeeping genes are widely used as internal controls in a variety of study types, including real time RT-PCR, microarrays, Northern analysis and RNase protection assays. However, even commonly used housekeeping genes may vary in stability depending on the cell type or disease being studied. Thus, it is necessary to identify additional housekeeping-type genes that show sample-independent stability. Here, we used statistical analysis to examine a large human microarray database, seeking genes that were stably expressed in various tissues, disease states and cell lines. We further selected genes that were expressed at different levels, because reference and target genes should be present in similar copy numbers to achieve reliable quantitative results. Real time RT-PCR amplification of three newly identified reference genes, CGI-119, CTBP1 and GOLGAl, alongside three well-known housekeeping genes, B2M, GAPD, and TUBB, confirmed that the newly identified genes were more stably expressed in individual samples with similar ranges. These results collectively suggest that statistical analysis of microarray data can be used to identify new candidate housekeeping genes showing consistent expression across tissues and diseases. Our analysis identified three novel candidate housekeeping genes (CGI-119, GOLGA1, and CTBP1) that could prove useful for normalization across a variety of RNA-based techniques.