Whole-genome sequence assemblies are now available for seven different animals, including nematode worms, mice and humans. Comparative genome analyses reveal a surprising constancy in genetic content: vertebrate genomes have only about twice the number of genes that invertebrate genomes have, and the increase is primarily due to the duplication of existing genes rather than the invention of new ones. How, then, has evolutionary diversity arisen? Emerging evidence suggests that organismal complexity arises from progressively more elaborate regulation of gene expression.

/pdf/transcription-regulation-and-animal-diversity-2qymt97v7s.pdf

Transcription regulation and animal diversity

Comparisons of orthologous genomic DNA sequences can be used to characterize regions that have been subject to purifying selection and are enriched for functional elements. We here present the results of such an analysis on an alignment of sequences from 29 mammalian species. The alignment captures ∼3.9 neutral substitutions per site and spans ∼1.9 Mbp of the human genome. We identify constrained elements from 3 bp to over 1 kbp in length, covering ∼5.5% of the human locus. Our estimate for the total amount of nonexonic constraint experienced by this locus is roughly twice that for exonic constraint. Constrained elements tend to cluster, and we identify large constrained regions that correspond well with known functional elements. While constraint density inversely correlates with mobile element density, we also show the presence of unambiguously constrained elements overlapping mammalian ancestral repeats. In addition, we describe a number of elements in this region that have undergone intense purifying selection throughout mammalian evolution, and we show that these important elements are more numerous than previously thought. These results were obtained with Genomic Evolutionary Rate Profiling (GERP), a statistically rigorous and biologically transparent framework for constrained element identification. GERP identifies regions at high resolution that exhibit nucleotide substitution deficits, and measures these deficits as “rejected substitutions.” Rejected substitutions reflect the intensity of past purifying selection and are used to rank and characterize constrained elements. We anticipate that GERP and the types of analyses it facilitates will provide further insights and improved annotation for the human genome as mammalian genome sequence data become richer.

/pdf/distribution-and-intensity-of-constraint-in-mammalian-2s2sr0y23z.pdf

Distribution and intensity of constraint in mammalian genomic sequence

In a previous paper we reported the successful use of graph coloring techniques for doing global register allocation in an experimental PL/I optimizing compiler. When the compiler cannot color the register conflict graph with a number of colors equal to the number of available machine registers, it must add code to spill and reload registers to and from storage. Previously the compiler produced spill code whose quality sometimes left much to be desired, and the ad hoe techniques used took considerable amounts of compile time. We have now discovered how to extend the graph coloring approach so that it naturally solves the spilling problem. Spill decisions are now made on the basis of the register conflict graph and cost estimates of the value of keeping the result of a computation in a register rather than in storage. This new approach produces better object code and takes much less compile time.

/pdf/register-allocation-spilling-via-graph-coloring-4o9u9dupfm.pdf

Register allocation & spilling via graph coloring

Microarray studies have shown recently that microbial infection leads to extensive changes in the Drosophila gene expression programme. However, little is known about the control of most of the fly immune-responsive genes, except for the antimicrobial peptide (AMP)-encoding genes, which are regulated by the Toll and Imd pathways. Here, we used oligonucleotide microarrays to monitor the effect of mutations affecting the Toll and Imd pathways on the expression programme induced by septic injury in Drosophila adults. We found that the Toll and Imd cascades control the majority of the genes regulated by microbial infection in addition to AMP genes and are involved in nearly all known Drosophila innate immune reactions. However, we identified some genes controlled by septic injury that are not affected in double mutant flies where both Toll and Imd pathways are defective, suggesting that other unidentified signalling cascades are activated by infection. Interestingly, we observed that some Drosophila immune-responsive genes are located in gene clusters, which often are transcriptionally co-regulated.

/pdf/the-toll-and-imd-pathways-are-the-major-regulators-of-the-58d8ygbtfc.pdf

The Toll and Imd pathways are the major regulators of the immune response in Drosophila

It is widely assumed that the key rate-limiting step in gene activation is the recruitment of RNA polymerase II (Pol II) to the core promoter. Although there are well-documented examples in which Pol II is recruited to a gene but stalls, a general role for Pol II stalling in development has not been established. We have carried out comprehensive Pol II chromatin immunoprecipitation microarray (ChIP-chip) assays in Drosophila embryos and identified three distinct Pol II binding behaviors: active (uniform binding across the entire transcription unit), no binding, and stalled (binding at the transcription start site). The notable feature of the approximately 10% genes that are stalled is that they are highly enriched for developmental control genes, which are either repressed or poised for activation during later stages of embryogenesis. We propose that Pol II stalling facilitates rapid temporal and spatial changes in gene activity during development.

/pdf/rna-polymerase-stalling-at-developmental-control-genes-in-3krheiq0gw.pdf

RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo.

Metazoan genomes contain vast tracts of cis-regulatory DNA that have been identified typically through tedious functional assays. As a result, it has not been possible to uncover a cis-regulatory code that links primary DNA sequences to gene expression patterns. In an initial effort to determine whether coordinately regulated genes share a common "grammar," we have examined the distribution of Dorsal recognition sequences in the Drosophila genome. Dorsal is one of the best-characterized sequence-specific transcription factors in Drosophila. The homeobox gene zerknullt (zen) is repressed directly by Dorsal, and this repression is mediated by a 600-bp silencer, the ventral repression element (VRE), which contains four optimal Dorsal binding sites. The arrangement and sequence of the Dorsal recognition sequences in the VRE were used to develop a computational algorithm to search the Drosophila genome for clusters of optimal Dorsal binding sites. There are 15 regions in the genome that contain three or more optimal sites within a span of 400 bp or less. Three of these regions are associated with known Dorsal target genes: sog, zen, and Brinker. The Dorsal binding cluster in sog is shown to mediate lateral stripes of gene expression in response to low levels of the Dorsal gradient. Two of the remaining 12 clusters are shown to be associated with genes that exhibit asymmetric patterns of expression across the dorsoventral axis. These results suggest that bioinformatics can be used to identify novel target genes and associated regulatory DNAs in a gene network.

https://www.zoology.ubc.ca/~bio463/pdf/763.pdf

Genome-wide analysis of clustered Dorsal binding sites identifies putative target genes in the Drosophila embryo.

Bioinformatics methods have identified enhancers that mediate restricted expression in the Drosophila embryo. However, only a small fraction of the predicted enhancers actually work when tested in vivo. In the present study, co-regulated neurogenic enhancers that are activated by intermediate levels of the Dorsal regulatory gradient are shown to contain several shared sequence motifs. These motifs permitted the identification of new neurogenic enhancers with high precision: five out of seven predicted enhancers direct restricted expression within ventral regions of the neurogenic ectoderm. Mutations in some of the shared motifs disrupt enhancer function, and evidence is presented that the Twist and Su(H) regulatory proteins are essential for the specification of the ventral neurogenic ectoderm prior to gastrulation. The regulatory model of neurogenic gene expression defined in this study permitted the identification of a neurogenic enhancer in the distant Anopheles genome. We discuss the prospects for deciphering regulatory codes that link primary DNA sequence information with predicted patterns of gene expression.

/pdf/a-regulatory-code-for-neurogenic-gene-expression-in-the-3ibw0e59pc.pdf

A regulatory code for neurogenic gene expression in the Drosophila embryo

A floating point processing system which uses a multiplier unit and an adder unit to perform floating point division and square root operations using both a conventional and a modified form of the Newton-Raphson method. The modified form of the Newton-Raphson method is used in place of the final iteration of the conventional Newton-Raphson so as to compute high precision approximated results with a substantial improvement in speed. The invention computes approximated results faster and simplifies hardware requirements because no multiplications of numbers of the precision of the result are required.

Floating point arithmetic unit using modified Newton-Raphson technique for division and square root

An analysis is given for optimizing run-time range checks in regions of high execution frequency. These optimizations are accomplished using strength reduction, code motion and common subexpression elimination. Test programs, using the above optimizations, are used to illustrate run-time improvements.

Optimization of range checking

We present division and square root algorithm for calculations with more bits than are handled by the floating-point hardware. These algorithms avoid the need to multiply two high-precision numbers, speeding up the last iteration by as much as a factor of 10. We also show how to produce the floating-point number closest to the exact result with relatively few additional operations.

/pdf/high-precision-division-and-square-root-38ejbwzerw.pdf

Peter Markstein

Papers

Genome-wide analysis of clustered Dorsal binding sites identifies putative target genes in the Drosophila embryo.

A regulatory code for neurogenic gene expression in the Drosophila embryo

Floating point arithmetic unit using modified Newton-Raphson technique for division and square root

Optimization of range checking

High-precision division and square root