Biomedical subjects

R J Britten

Publications and source records attributed to R J Britten.

At least 19 recordsLinked to original sources

PubMed1998

Underlying assumptions of developmental models.

These 10 obvious propositions make a model of the specification of form, intended to expose underlying assumptions of developmental biology for examination and future experimentation. (I) The control of development is by means of local interactions, rather than global control mechanisms. (II) A macromolecule near a specific site will bind by mass action. (III) Starting with a precursor cell, all cells are assembled automatically by specifically binding macromolecules. (IV) At the surface of cells are specific adhesion sites that determine how all cells bind to each other. (V) An organism will assemble automatically from parts (macromolecules, structures, and cells) specified by nuclear control factors. (VI) The nuclear control factors in each cell are from precursor cells and factors derived by signaling from other cells. (VII) The macromolecules that determine specific binding, cell adhesion, and signaling are controlled by nuclear control factors, and in a grand feedback the cell adhesion and signaling systems determine the nuclear factor patterns. (VIII) The embryonic precursor cells for organs, termed "precursor groups," are linked by adhesion and signaling relationships. (IX) The precursor groups include precursors for regions of an organ and boundary cells between regions having few cell types, growing without additional specific cell-to-cell relationships. (X) Organs are held together by cell adhesion in functional relationships. Thus the form and function of the organism is specified entirely by local control mechanisms. Without global control systems, information for form is in the genes for structural proteins, adhesion molecules, control factors, signaling molecules, and their control regions.

Animals↗

PubMed1998

Precise sequence complementarity between yeast chromosome ends and two classes of just-subtelomeric sequences.

The terminal regions (last 20 kb) of Saccharomyces cerevisiae chromosomes universally contain blocks of precise sequence similarity to other chromosome terminal regions. The left and right terminal regions are distinct in the sense that the sequence similarities between them are reverse complements. Direct sequence similarity occurs between the left terminal regions and also between the right terminal regions, but not between any left ends and right ends. With minor exceptions the relationships range from 80% to 100% match within blocks. The regions of similarity are composites of familiar and unfamiliar repeated sequences as well as what could be considered "single-copy" (or better "two-copy") sequences. All terminal regions were compared with all other chromosomes, forward and reverse complement, and 768 comparisons are diagrammed. It appears there has been an extensive history of sequence exchange or copying between terminal regions. The subtelomeric sequences fall into two classes. Seventeen of the chromosome ends terminate with the Y' repeat, while 15 end with the 800-nt "X2" repeats just adjacent to the telomerase simple repeats. The just-subterminal repeats are very similar to each other except that chromosome 1 right end is more divergent.

Base Sequence↗

PubMed1998

A gravitational diffusion model without dark matter.

In this model, without dark matter, the flat rotation curves of galaxies and the mass-to-light ratios of clusters of galaxies are described quantitatively. The hypothesis is that the agent of gravitational force is propagated as if it were scattered with a mean free path of approximately 5 kiloparsecs. As a result, the force between moderately distant masses, separated by more than the mean free path, diminishes as the inverse first power of the distance, following diffusion equations, and describes the flat rotation curves of galaxies. The force between masses separated by <1 kiloparsec diminishes as the inverse square of distance. The excess gravitational force (ratio of 1/r:1/r2) increases with the scale of structures from galaxies to clusters of galaxies. However, there is reduced force at great distances because of the approximately 12 billion years that has been available for diffusion to occur. This model with a mean free path of approximately 5 kiloparsecs predicts a maximum excess force of a few hundredfold for objects the size of galactic clusters a few megaparsecs in size. With only a single free parameter, the predicted curve for excess gravitational force vs. size of structures fits reasonably well with observations from those for dwarf galaxies through galactic clusters. Under the diffusion model, no matter is proposed in addition to the observed baryons plus radiation and thus the proposed density of the universe is only a few percent of that required for closure.

Journal Article↗

PubMed1997

Mobile elements inserted in the distant past have taken on important functions.

Current evidence on the long-term evolutionary effect of insertion of sequence elements is reviewed. There are three criteria for inclusion of an example: (i) the element was inserted far in the past and thus the event is not a transient mutation; (ii) the element is a member of a large group of similar sequences; (iii) the element now serves a useful function. There are 21 examples from Drosophila, sea urchin, human and mouse genomes that meet these criteria. Taken together, these examples show that the insertion of sequence elements in the genome has been a significant source of regulatory variation in evolution.

Animals↗

PubMed1996

DNA sequence insertion and evolutionary variation in gene regulation.

Current evidence on the long-term evolutionary effect of insertion of sequence elements into gene regions is reviewed, restricted to cases where a sequence derived from a past insertion participates in the regulation of expression of a useful gene. Ten such examples in eukaryotes demonstrate that segments of repetitive DNA or mobile elements have been inserted in the past in gene regions, have been preserved, sometimes modified by selection, and now affect control of transcription of the adjacent gene. Included are only examples in which transcription control was modified by the insert. Several cases in which merely transcription initiation occurred in the insert were set aside. Two of the examples involved the long terminal repeats of mammalian endogenous retroviruses. Another two examples were control of transcription by repeated sequence inserts in sea urchin genomes. There are now six published examples in which Alu sequences were inserted long ago into human gene regions, were modified, and now are central in control/enhancement of transcription. The number of published examples of Alu sequences affecting gene control has grown threefold in the last year and is likely to continue growing. Taken together, all of these examples show that the insertion of sequence elements in the genome has been a significant source of regulatory variation in evolution.

Animals↗

PubMed1996

Sea urchin genes expressed in activated coelomocytes are identified by expressed sequence tags. Complement homologues and other putative immune response genes suggest immune system homology within the deuterostomes.

To identify some of the genes expressed in LPS-activated coelomocytes, we sequenced randomly chosen clones from a directionally constructed cDNA library to produce a set of expressed sequence tags (ESTs). Deduced amino acid sequences from 307 ESTs were compared with known protein sequences in GenBank, and significant matches to approximately 30% of the clones were identified. Eighty-nine clones matched to 55 different proteins, including several putative immune effector proteins. In this work, we show the first identification of an invertebrate homologue of a vertebrate C component. Another EST matches to several short consensus repeats that are characteristic of a variety of proteins, including CR/regulatory proteins and clotting factors. Additional putative immune effector genes include 1) a Kazal-type protease inhibitor that may function to inactivate bacterial proteases, 2) a C-type lectin similar to echinoidin, and 3) a serine protease with similarities to thrombin, elastase, haptoglobin, and plasmin. Other EST categories include 1) cell surface proteins and receptors, 2) proteins involved in signaling systems, 3) lysosomal and secreted proteins, 4) cytoskeletal and cytoskeletal modifying proteins, 5) general cell function proteins, 6) proteins with unknown function, and 7) ESTs without significant matches, 25 with open reading frames. Many of the ESTs identified in this study represent the types of genes expected to be used in lower deuterostome immune functions.

Amino Acid Sequence↗

PubMed1996

Cases of ancient mobile element DNA insertions that now affect gene regulation.

There is no doubt that mobile elements are a major source of mutation and that in many cases insertions cause changes in the expression of genes, but a question remains open. What has the long-term effect of these processes been? The data collected here show that in many eukaryotes, segments of repetitive DNA (mobile elements) that have been inserted in the past in regions of many eukaryote genes have been preserved by selection and now affect the transcriptional control of these specific genes. At least five of the examples are considered to be solid cases demonstrating this history. There are a number of significant but less compelling sets of evidence that support the concept that the insertion of mobile elements in gene regions could be a major source of regulatory variation in evolution.

Amylases↗

PubMed1995

A multimerizing transcription factor of sea urchin embryos capable of looping DNA.

SpGCF1 is a recently cloned sea urchin transcription factor that recognizes target sites in several different sea urchin genes. We find that in gel-shift experiments this factor is able to multimerize. A quantitative simulation of the gel-shift results suggests that SpGCF1 molecules that are bound to DNA target sites may also bind to one another, thus associating several DNA probe molecules. SpGCF1 might therefore be able to loop DNA molecules bearing its target sites at distant locations. We demonstrate this prediction by electron microscopy, and using the well-characterized cis-regulatory domain of the CyIIIa cytoskeletal actin gene, we show that the loop conformations predicted from the known SpGCF1 target site locations are actually formed in vitro. We speculate that the multimerization of this factor in vivo may function to bring distant regions of extended regulatory domains into immediate proximity so that they can interact with one another.

Actins↗

PubMed1995

Active gypsy/Ty3 retrotransposons or retroviruses in Caenorhabditis elegans.

A gypsy/Ty3-class retrotransposon (Cer1) is integrated in the DNA of Caenorhabditis elegans chromosome III. It is 8865 nt in length and has 492-nt long terminal repeats that are identical in DNA sequence. There is an exceptionally long (6819 nt) open reading frame uninterrupted by frame-shift mutations in the period since the insertion, which must therefore have been rather recent. Alignment with other gypsy-class elements and with retroviruses indicates that an env gene occupies the 3' 1.2 kb of the open reading frame. A search through GenBank has uncovered two additional gypsy-class elements from C. elegans that are very closely related in DNA sequence to this insert and are transcribed. Since gypsy of Drosophila has been shown to be an infectious element, it is possible that retrovirus-like gypsy elements are active in C. elegans.

Animals↗

PubMed1995

SpGCF1, a sea urchin embryo DNA-binding protein, exists as five nested variants encoded by a single mRNA.

Several Strongylocentrotus purpuratus gene cis-regulatory regions contain asymmetric C4 sequences which are core elements of target sites for a specific DNA-protein interaction. Blastula stage nuclear extract contains five proteins which specifically bind to these target sites, resulting in a characteristic pattern of complexes in gel mobility shift assays. We used automated affinity chromatography to purify a protein which binds to these sites and have isolated the corresponding cDNA. This protein, SpGCF1, is a novel sea urchin DNA-binding protein with no overall homology to proteins reported in the databases currently available. The DNA-binding domain of this protein was identified by a deletion analysis. As demonstrated both for protein translated in vitro and for bacterial protein expressed from a cDNA clone, a single SpGCF1 mRNA serves as a template for the synthesis of five DNA-binding polypeptides. We show that these five polypeptides are most likely produced by differential usage of a nested set of AUG start codons in the SpGCF1 cDNA and thus contain variable amounts of a proline-rich N-terminal domain. Since proline-rich regions often serve as transcriptional activation domains, the five SpGCF1 proteins apparently possess different "activation potentials."

Amino Acid Sequence↗

PubMed1995

Developmental utilization of SpP3A1 and SpP3A2: two proteins which recognize the same DNA target site in several sea urchin gene regulatory regions.

SpP3A1 and SpP3A2 are DNA-binding proteins that interact specifically with the same target sites in the regulatory domains of the Strongylocentrotus purpuratus CyIIIa gene and also of several other known genes. In this work we used antibodies raised against recombinant P3A1 and P3A2 to quantitate these transcription factors in eggs and in the nuclear compartments of embryos of various stages. Both proteins are present in unfertilized eggs, and both enter the embryonic nuclei early in development, but only P3A2 remains present in nuclei at functional concentrations beyond the early gastrula stage. Combined with earlier measurements of P3A site binding at cleavage stages, these measurements show that P3A1 would be replaced by P3A2 at target sites in genes regulated by these factors.

Animals↗

PubMed1995

Gypsy/Ty3-class retrotransposons integrated in the DNA of herring, tunicate, and echinoderms.

Eight new examples of retrotransposons of the Gypsy/Ty3 class have been identified in marine species. A 525-nt pol gene-coding region was amplified using degenerate primers from highly conserved regions and has extended the range of recognition of Gypsy/Ty3 far beyond those previously known. The following matrix shows the percentage AA divergence of the translations of this segment of the pol gene coding region. [table: see text] The underlines separate three groups of retrotransposons that can be recognized on the basis of this amino acid sequence. The new upper group shows surprising amino acid sequence similarity among members from the DNA of herring, sea urchin, starfish, and a tunicate. For example, the herring element differs by only 41% from the Ciona element and 46% from the sea urchin element. The group between the lines includes members close to previously known elements (marked by asterisks) and has so far been found only in sea urchins. The two upper groups differ from each other by 55-60% and yet members of both groups (e.g., Spr1 and Spr2) are integrated into the DNA of one species--S. purpuratus. Below the lower underline is listed the only known representative of a very distant group, which occurs in starfish DNA. In spite of large divergence, amino acid sequence comparisons indicate that all of the elements shown in the array are members of the LTR-containing class of retrotransposons that includes Gypsy of Drosophila and Ty3 of yeast. Of all known mobile elements this class shows the closest sequence similarity to retroviruses and has the same arrangement of genes as simpler retroviruses.

Amino Acid Sequence↗

PubMed1995

Phylogeny, rates of evolution, and patterns of codon usage among sea urchin retroviral-like elements, with implications for the recognition of horizontal transfer.

Phylogenetic relationships, rates of evolution, and codon usage were investigated in a family of retrotransposons (SURL elements) found in echinoids. The phylogeny of SURL element reverse transcriptase sequences from 10 echinoid species clearly shows the phylogenetic signature of the host taxa as well as paralogous sequences that diverged prior to speciation events. Two subfamilies (1 and 5) of SURL element reverse transcriptase sequences are recognized that diverged prior to the radiation of the Echinometridae. Comparisons of synonymous versus nonsynonymous substitutions indicate that SURL elements have been active in echinoid genomes and have evolved under purifying selection for millions of years. Rates of synonymous substitution for reverse transcriptase are similar to rates of single-copy DNA evolution and to rates of synonymous substitution for the H3 and H4 histone genes, contradicting the assumption that rates of evolution are accelerated in retrotransposons. Finally, codon usage in SURL elements is biased for codons ending in A or U relative to 42 sea urchin nuclear genes. Biased codon usage is sometimes cited as evidence for horizontal transfer, but in the case of SURL elements this bias occurs in spite of a long history of vertical transmission rather than because of horizontal transfer.

Animals↗

PubMed1995

SpZ12-1, a negative regulator required for spatial control of the territory-specific CyIIIa gene in the sea urchin embryo.

The CyIIIa cytoskeletal actin gene of the sea urchin Strongylocentrotus purpuratus is activated in late cleavage and expressed exclusively in the aboral ectoderm territory of the embryo. Previous gene transfer studies defined a 2.3 kb cis-regulatory region that is necessary and sufficient for correct temporal and spatial expression of a CyIIIa.CAT fusion gene. In this paper, a negative regulatory element within this region was identified that is required for repression of the CyIIIa gene in skeletogenic mesenchyme cells. The repression mediated by this regulatory element takes place after initial territorial specification. A cDNA clone encoding a DNA-binding protein with twelve Zn fingers (SpZ12-1) was isolated by probing an expression library with this cis-element. Deletion analysis of the SpZ12-1 protein confirmed that a DNA-binding domain is located within the Zn finger region. SpZ12-1 is the only DNA-binding protein in embryo nuclear extract that interacts with the specific cis-target sites required for repression of CyIIIa.CAT in skeletogenic mesenchyme and is likely to be the trans factor that mediates this repression.

Actins↗

PubMed1995

Cis-regulatory control of the SM50 gene, an early marker of skeletogenic lineage specification in the sea urchin embryo.

The SM50 gene encodes a minor matrix protein of the sea urchin embryo spicule. We carried out a detailed functional analysis of a cis-regulatory region of this gene, extending 440 bp upstream and 120 bp downstream of the transcription start site, that had been shown earlier to confer accurate skeletogenic expression of an injected expression vector. The distal portion of this fragment contains elements controlling amplitude of expression, while the region from -200 to +105 contains spatial control elements that position expression accurately in the skeletogenic lineages of the embryo. A systematic mutagenesis analysis of this region revealed four adjacent regulatory elements, viz two copies of a positively acting sequence (element D) that are positioned just upstream of the transcription start site; an indispensable spatial control element (element C) that is positioned downstream of the start site; and further downstream, a second positively acting sequence (element A). We then constructed a series of synthetic expression constructs. These contained oligonucleotides representing normal and mutated versions of elements D, C, and A, in various combinations. We also changed the promoter of the SM50 gene from a TATA-less to a canonical TATA box form, without any effect on function. Perfect spatial regulation was also produced by a final series of constructs that consisted entirely of heterologous enhancers from the CyIIIa gene, the SV40 early promoter, and synthetic D, C, and A elements. We demonstrate that element C exercises the primary spatial control function of the region we analyzed. We term this a 'locator' element. This differs from conventional 'tissue-specific enhancers' in that while it is essential for expression, it has no transcriptional activity on its own, and it requires other, separable, positive regulatory elements for activity. In the normal configuration these ancillary positive functions are mediated by elements A and D. Only positively acting control elements were observed in the SM50 regulatory domain throughout this analysis.

Animals↗

PubMed1995

Maternal and embryonic provenance of a sea urchin embryo transcription factor, SpZ12-1.

SpZ12-1 is a zinc-finger transcription factor. Previous work has indicated that this factor functions late in embryogenesis as a spatial transcriptional repressor. We show here that this factor is present in significant quantities even in unfertilized egg cytoplasm, and in similar quantities in mesenchyme blastula-stage embryo cytoplasm. Taken together with earlier measurements of Calzone and associates, our observations indicate that SpZ12-1 enters the embryonic nuclei between late cleavage and mesenchyme blastula stages. A low-prevalence mRNA encoding SpZ12-1 is also present throughout development. Translation of this mRNA could, however, easily account for the complete complement of SpZ12-1 protein in the embryo, as estimated from its DNA binding activity. SpZ12-1 probably functions at several developmental stages and is evidently of both maternal and embryonic provenance.

Animals↗

PubMed1994

Evolutionary selection against change in many Alu repeat sequences interspersed through primate genomes.

Mutations have been examined in the 1500 interspersed Alu repeats of human DNA that have been sequenced and are nearly full length. There is a set of particular changes at certain positions that rarely occur (termed suppressed changes) compared to the average of identical changes of identical nucleotides in the rest of the sequence. The suppressed changes occur in positions that are clustered together in what appear to be sites for protein binding. There is a good correlation of the suppression in different positions, and therefore the joint probability of absence of mutation at many pairs of such positions is significantly higher than that expected at random. The suppression of mutation appears to result from selection that is not due to requirements for Alu sequence replication. The implication is that hundreds of thousands of Alu sequences have sequence-dependent functions in the genome that are selectively important for primates. In a few known cases Alu inserts have been adapted to function in the regulation of gene transcription.

Animals↗

PubMed1994

Evidence that most human Alu sequences were inserted in a process that ceased about 30 million years ago.

The primate Alu interspersed repeats can be subdivided into classes on the basis of shared nucleotides at a set of diagnostic positions. Each of the classes of Alu sequences is apparently the result of past retrotransposition of transcripts of highly conserved class-specific source genes that differed from each other at the diagnostic positions. The nucleotides at the majority of positions are identical among the source genes and therefore were identical among all of the Alu sequences at the time of their insertion. These CONSBI (conserved before insertion) positions are useful because the changes that have occurred after insertion are recognizable and the divergence resulting from nucleotide substitutions, insertions, and deletions is informative. The divergence of Alu sequences at the CONSBI positions is a measure of the time since a class was inserted. The greatest majority of Alu sequences are in one class (identified as class II), and it is particularly suitable for such examination, since nearly full-length sequences are now known for nearly a thousand members of this class. The average divergence of class II members indicates that the class has an average age of about 40 million years. The distribution in divergence of class II accurately fits a sum of two Poisson distributions. The implication is that class II Alu sequences were derived from two massive past events of insertion of many Alu sequences. In this model the younger subset of class II sequences (corresponding to about 300,000 copies in the genome) has an average divergence of 5% at CONSBI positions. The older set of class II sequences (corresponding to about 150,000 genomic copies) has a 9% average divergence. Based on the drift rate of primate DNA sequences, the events of insertion probably occurred 30-50 million years ago. The goodness of the fit to the Poisson distribution indicates that no significant number of members of class II have been inserted since 30 million years ago.

Animals↗