Almost all human genes resulted from ancient duplication

Results of protein sequence comparison at open criterion show a very large number of relationships that have, up to now, gone unreported. The relationships suggest many ancient events of gene duplication. It is well known that gene duplication has been a major process in the evolution of genomes. A collection of human genes that have known functions have been examined for a history of gene duplications detected by means of amino acid sequence similarity by using BLASTp with an expectation of two or less (open criterion). Because the collection of genes in build 35 includes sets of transcript variants, all genes of known function were collected, and only the longest transcription variant was included, yielding a 13,298-member library called KGMV (for known genes maximum variant). When all lengths of matches are accepted, >97% of human genes show significant matches to each other. Many form matches with a large number of other different proteins, showing that most genes are made up from parts of many others as a result of ancient events of duplication. To support the use of the open criterion, all of the members of the KGMV library were twice replaced with random protein sequences of the same length and average composition, and all were compared with each other with BLASTp at expectation two or less. The set of matches averaged 0.35% of that observed for the KGMV set of proteins

Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA

A method is described for microinjection of cloned DNA into the zygote nucleus of Lytechinus variegatus. Eggs of this species are unusually transparent, facilitating visual monitoring of the injection process. The initial fate of injected DNA fragments appears similar to that observed earlier for exogenous DNA injected into unfertilized egg cytoplasm. Thus after end-to-end ligation, it is replicated after a lag of several hours to an extent indicating that it probably participates in most of the later rounds of DNA synthesis undergone by the host cell genomes during cleavage. The different consequences of nuclear versus cytoplasmic injection are evident at advanced larval stages. Larvae descendant from eggs in which exogenous DNA was injected into the nuclei are four times more likely (32% versus 8%) to retain this DNA in cell lineages that replicate very extensively during larval growth, i.e. the lineages contributing to the imaginal rudiment, and thus to display greatly enhanced contents of the exogenous DNA. Similarly, 36% of postmetamorphic juveniles from a nuclear injection sample retained the exogenous DNA sequences, compared to 12% of juveniles from a cytoplasmic injection sample. However, the number of copies of the exogenous DNA sequences retained per average genome in postmetamorphic juveniles was usually less than 0.1 (range 0.05-50), and genome blot hybridizations indicate that these sequences are organized as integrated, randomly oriented, end-to-end molecular concatenates. It follows that only a small fraction of the cells of the average juvenile usually retains the exogenous sequences. Thus, even when introduced by nuclear microinjection, the stable incorporation of exogenous DNA in the embryo occurs in a mosaic fashion, although in many recipients the DNA enters a wider range of cell lineages than is typical after cytoplasmic injection. Nuclear injection would probably be the route of choice for studies of exogenous DNA function in the postembryonic larval rudiment

The oral-aboral axis of a sea urchin embryo is specified by first cleavage

Several lines of evidence suggest that the oral-aboral axis in Strongylocentrotus purpuratus embryos is specified at or before the 8-cell stage. Were the oral-aboral axis specified independently of the first cleavage plane, then a random association of this plane with the blastomeres of the four embryo quadrants in the oral-aboral plane (viz. oral, aboral, right and left) would be expected. Lineage tracer dye injection into one blastomere at the 2-cell stage and observation of the resultant labeling patterns demonstrates instead a strongly nonrandom association. In at least ninety percent of cases, the progeny of the aboral blastomeres are associated with those of the left lateral blastomeres and the progeny of the oral blastomeres with the right lateral ones, respectively. Thus, ninety percent of the time the oral pole of the future oral-aboral axis lies 45 degrees clockwise from the first cleavage plane as viewed from the animal pole. The nonrandom association of blastomeres after labeling of the 2-cell stage implies that there is a mechanistic relation between axis specification and the positioning of the first cleavage plane

Macromere cell fates during sea urchin development

This paper examines the cell lineage relationships and cell fates in embryos of the sea urchin Strongylocentrotus purpuratus leading to the various cell types derived from the definitive vegetal plate territory or the veg_2 tier of cells. These cell types are gut, pigment cells, basal cells and coelomic pouches. They are cell types that constitute embryonic structures through cellular migration or rearrangement unlike the relatively non-motile ectoderm cell types. For this analysis, we use previous knowledge of lineage to assign macromeres to one of four types: VOM, the oral macromere; VAM, the aboral macromere, right and left VLM, the lateral macromeres. Each of the four macromeres contributes progeny to all of the cell types that descend from the definitive vegetal plate. Thus in the gut each macromere contributes to the esophagus, stomach and intestine, and the stripe of labeled cells descendant from a macromere reflects the re-arrangement of cells that occurs during archenteron elongation. Pigment cell contributions exhibit no consistent pattern among the four macromeres, and are haphazardly distributed throughout the ectoderm. Gut and pigment cell contributions are thus radially symmetrical. In contrast, the VOM blastomere contributes to both of the coelomic pouches while the other three macromeres contribute to only one or the other pouch. The total of the macromere contribution amounts to 60% of the cells constituting the coelomic pouches

Negative spatial regulation of the lineage specific CyIIIa actin gene in the sea urchin embryo

The CyIIIa·CAT fusion gene was injected into Strongylocentrotus purpuratus eggs, together with excess ligated competitor sequences representing subregions of the CyIIIa regulatory domain. In this construct, the chloramphenicol acetyltransferase (CAT) reporter gene is placed under the control of the 2300 nucleotide upstream regulatory domain of the lineage-specific CyIIIa cytoskeletal actin gene. CAT mRNA was detected by in situ hybridization in serial sections of pluteus stage embryos derived from the injected eggs. When carrier DNA lacking competitor CyIIIa fragments was coinjected with CyIIIa.CAT, CAT mRNA was observed exclusively in aboral ectoderm cells, i.e. the territory in which the CyIIIa gene itself is normally expressed (as also reported by us previously). The same result was obtained when five of seven different competitor subfragments bearing sites of DNA-protein interaction were coinjected. However, coinjection of excess quantities of either of two widely separated, nonhomologous fragments of the CyIIIa regulatory domain produced a dramatic ectopic expression of CAT mRNA in the recipient embryos. CAT mRNA was observed in gut, mesenchyme cells and oral ectoderm in these embryos. We conclude that these fragments contain regulatory sites that negatively control spatial expression of the CyIIIa gene

Competitive titration in living sea urchin embryos of regulatory factors required for expression of the CyIIIa actin gene

Previous studies have located some twenty distinct sites within the 2.3 kb 5' regulatory domain of the sea urchin CyIIIa cytoskeletal actin gene, where there occur in vitro high-specificity interactions with nuclear DNA-binding proteins of the embryo. This gene is activated in late cleavage, exclusively in cells of the aboral ectoderm cell lineages. In this study, we investigate the functional importance in vivo of these sites of DNA-protein interaction. Sea urchin eggs were coinjected with a fusion gene construct in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene is under the control of the entire CyIIIa regulatory domain, together with molar excesses of one of ten nonoverlapping competitor subfragments of this domain, each of which contains one or a few specific site(s) of interaction. The exogenous excess binding sites competitively titrate the available regulatory factors away from the respective sites associated with the CyIIIa.CAT reporter gene. This provides a method for detecting in vivo sites within the regulatory domain that are required for normal levels of expression, without disturbing the structure of the regulatory domain. We thus identify five nonoverlapping regions of the regulatory DNA that apparently function as binding sites for positively acting transcriptional regulatory factors. Competition with a subfragment bearing an octamer site results in embryonic lethality. We find that three other sites display no quantitative competitive interference with CyIIIa.CAT expression, though as shown in the accompanying paper, two of these sites are required for control of spatial expression. We conclude that the complex CyIIIa regulatory domain must assess the state of many distinct and individually necessary interactions in order to properly regulate CyIIIa transcriptional activity in development

Speed and Accuracy of Static Image Discrimination by Rats

When discriminating dynamic noisy sensory signals, human and primate subjects achieve higher accuracy when they take more time to decide, an effect attributed to accumulation of evidence over time to overcome neural noise. We measured the speed and accuracy of twelve freely behaving rats discriminating static, high contrast photographs of real-world objects for water reward in a self-paced task. Response latency was longer in correct trials compared to error trials. Discrimination accuracy increased with response latency over the range of 500-1200ms. We used morphs between previously learned images to vary the image similarity parametrically, and thereby modulate task difficulty from ceiling to chance. Over this range we find that rats take more time before responding in trials with more similar stimuli. We conclude that rats' perceptual decisions improve with time even in the absence of temporal information in the stimulus, and that rats modulate speed in response to discrimination difficulty to balance speed and accuracy