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L Kedes

Publications and source records attributed to L Kedes.

142 records · Page 8Linked to original sources
PubMed1982

MAXAMIZE. A DNA sequencing strategy advisor.

The MAXAMIZE advisory system determines from user-provided restriction maps an optimal strategy to do nucleotide sequencing by methods involving end-labeled fragments. The maps may be either simple linear restriction maps of fragments or complex circular maps including restriction sites of a vector. The whole system is interactive and is written in the Genetic English language provided by the GENESIS System, a molecular genetics knowledge representation and manipulation package. In addition, MAXAMIZE provides bookkeeping facilities for sequencing and offers advise on how to verify the newly obtained sequence data.

Base Sequence↗
PubMed1982

GEL, a DNA sequencing project management system.

We have developed an automated system for management of DNA sequencing projects. The system, named GEL, can handle data from both random sequences and from fragments whose relative positions are known. The system is highly interactive, self-documenting, and forgiving; it is designed for use by computer-naive molecular biologists. An editor designed specifically for sequences allows simple entry of data. Special functions allow direct checking and immediate editing of paired readings of the same gel. Merging of new random fragment sequences into the project as a whole is semi-automated. The user is shown probable overlaps if they exist, and can edit either the sequences or the consensus. Heuristic approaches to limiting the kinds of searches made in the merging process reduces the problem of combinatoric data overload as sequencing projects grow large. Complete histories of all entries, editing changes, and generation of consensus sequences are automatically prepared.

Base Sequence↗
PubMed1982

GENESIS, a knowledge-based genetic engineering simulation system for representation of genetic data and experiment planning.

We have built a knowledge-based genetic engineering simulation system-- GENESIS-- capable of representing both domain-specific and general knowledge. Information is stored within a hierarchically-organized framework composed of structures called units. A series of sophisticated editors enables no-computer specialist molecular geneticists to construct a knowledge base through direct interaction with the computer. Three types of knowledge specific to the domain of molecular genetics, MAPS, sequences and RULES are discussed in detail with examples.

Animals↗
PubMed1982

Human cytoplasmic actin proteins are encoded by a multigene family.

We characterized nine human actin genes that we isolated (Engel et al., Proc. Natl. Acad. Sci. U.S.A. 78:4674-4678, 1981) from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and alpha-, beta-, and gamma-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria we show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken beta-actin cDNA. We conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.

Actins↗
PubMed1981

Orphons: dispersed genetic elements derived from tandem repetitive genes of eucaryotes.

We have discovered in animal cells a novel class of dispersed, solitary genetic elements derived from tandem multigene families. We refer to such displaced elements as orphons. Orphons arise from both protein-coding and non-protein-coding structural gene families, including those of histone and ribosomal genes. Southern transfer hybridization experiments, on DNA digested with enzymes that do not cut the major repeat unit of the family of genes of interest, reveal histone gene orphons in the sea urchin (Lytechinus pictus), ribosomal gene orphons in yeast (Saccharomyces cerevisiae) and ribosomal and H3 histone gene orphons in Drosophila melanogaster. There are more than 50 histone gene orphons in each sea urchin genome. Each of the five histone-coding regions has a number of orphons (5--20), in addition to the several hundred copies in the clusters that are not cut by Bam HI. Most such orphons appear to contain only one coding region. Nearly all sea urchin histone gene orphon loci are polymorphic in the population; no two individuals have the same sets of histone orphons for any coding region. An H3-coding orphon, pLpH30-1, was isolated from the genome of L. pictus. The H3 region homologous to the histone gene clusters is 1200 bases long and includes the complete early H3 gene and some surrounding histone DNA. THe orphon is flanked by at least 1.7 kb of 5' and 3 kb of 3' nonhistone DNA. These flanking sequences are each moderately repetitive in the genome and are not homologous to each other. The orphon differs less than 2% in base sequence from the analogous region of pLpC and only slightly more from pLpA (the major histone gene clones). DNA sequence analysis of the junctions between the H3 region and the immediate flanking nonhistone DNA reveals no evidence of repetitive or palindromic sequences.

Animals↗
PubMed1981

Isolation and characterization of human actin genes.

We have utilized cloned actin genes from Drosophila melanogaster and from chicken to isolate 12 actin gene fragments from a human DNA library. Each of these 12 clones was shown to contain actin coding regions by its ability to selectively hybridize to human actin mRNA as assayed by in vitro translation. The translation product was judged to be actin on the basis of its comigration with authentic actins when electrophoresed on one- and two-dimensional NaDodSO4/polyacrylamide gels and on the basis of its partial proteolysis products. Determination of the sizes and order of the fragments generated by restriction endonuclease digestion of each of these recombinant phages allows us to conclude that they are nonallelic and are from nonoverlapping regions of the genome. We have used these cloned human actin genes and the Drosophila and chicken actin gene clones to show that the human genome contains 25-30 EcoRI fragments homologous to actin genes and that, among three nonconsanguineous individuals tested, none of these fragments exhibit length or restriction-site polymorphism.

Actins↗
PubMed1981

Monocistronic transcription is the physiological mechanism of sea urchin embryonic histone gene expression.

We have examined histone gene expression during the early stages of sea urchin embryogenesis. The five histone genes expressed at that time are contained in tandem repetitive segments. It has been suggested that adjacent coding regions and their intervening spacer sequences are transcribed into large polycistronic messenger ribonucleic acid (RNA) precursors. We have subcloned into pBR322 deoxyribonucleic acid (DNA) sequences mapping either in the coding region, the 5' spacer, or the 3' spacer of the H2B histone gene. These clones were used to produce radioiodinated hybridization probes. We measured the steady-state quantity of H2B messenger RNA as well as spacer-specific RNA in the total RNA from embryos taken at various stages of development from fertilization to hatching of blastulae (0 to 22 h post-fertilization). Small amounts of RNA hybridizing to both spacer probes could be found. However, we show that these RNAs form mismatched hybrids with the spacer DNA and therefore cannot originate from the spacers present in the histone genes. We conclude that there is no detectable transcription of the spacer regions on either side of the H2B histone gene. The detection limit for RNA complementary to the 5' spacer sequence corresponds to a maximum of about three RNA molecules per cell, an amount shown to be far less than the projected steady-state pool size of a putative polycistronic transcript, if such a precursor were to be the obligatory transcript of the histone genes. (This conclusion was derived by using the known rates of production of H2B mRNA throughout early development [R. E. Maxson and F. H. Wilt, Dev. Biol., in press].) The physiologically relevant transcript of the histone genes in early development is therefore monocistronic and probably identical to the messenger RNA itself.

Animals↗
PubMed1978

Sea urchin nuclei use RNA polymerase II to transcribe discrete histone RNAs larger than messengers.

RNA transcribed in isolated sea urchin nuclei and assayed by hybridization to histone genes cloned in E. coli contains sequences homologous to each of the five histone genes. Histone RNA is synthesized exclusively from the same DNA strand which is the template in vivo. Synthesis of the histone gene transcripts is sensitive to alpha-amanitin concentrations which inhibit RNA polymerase II activity. The fraction of histone RNA synthesized in vitro is comparable at two developmental stages to the fraction synthesized in vivo. The nuclear histone transcripts contain sequences homologous to spacer DNA regions present between the coding regions of the 6500 base pair (bp) histone gene repeat unit. The transcription of spacer sequences was demonstrated by hybridization of the nuclear transcripts to subcloned spacer DNA. Although the bulk of the RNA transcripts are greater than 2000 bases long, the histone-specific transcripts are of discrete sizes ranging from 100 bases to about 1100 bases long. Each histone gene hybridizes with at least one of the larger transcripts and with a different subset of smaller RNAs. We do not detect any giant polycistronic transcript spanning the entire histone repeat unit.

Amanitins↗
PubMed1975

Individual histone messenger RNAs: identification by template activity.

Newly synthesized polysomal messenger RNAs from cleavage stage embryos of the sea urchin Arbacia punctulata and Lytechinus pictus that contain putative histone mRNAs have been fractionated on 6% polyacrylamide slab gels. At least 8 RNA species with unique electrophoretic mobilities have been recognized. The complex of RNAs has been eluted from the gels in three groups, A, B, and C, in increasing order of mobility. The template activity of the three fractions and the unfractionated starting material was examined in the mouse Krebs II ascites tumor cell-free protein synthesizing system. The unfractionated messenger complex programs the synthesis of proteins that co-electrophorese exclusively with sea urchin histones in both sodium dodecyl sulfate and acid urea gel systems. The products of in vitro protein systhesis stimulated by the individual polyacrylamide gel RNA fractions were similarly examined. Each stimulated protein synthesis and was enriched for specific histone templates. We conclude that RNA fraction A is template for histone f1, C is template for histone f2a1, and B serves as template for f2b, f2a2, and f3 histones. A minor degree of contamination of the A and B RNA fractions was obvious from the production of other histones by each template. The co-electrophoresis of specific template activity with specific radiolabeled RNAs supports the concept that most or all of the labeled RNAs are indeed themselves the histone mRNAs.

Animals↗
PubMedDate not supplied

An unusual transposon with long terminal inverted repeats in the sea urchin Strongylocentrotus purpuratus.

A 3-kilobase DNA segment characteristic of a transposable element was found within a histone H2B pseudogene in a higher eukaryote, the sea urchin Stronglyocentrotus purpuratus. The inserted segment (TU1) is flanked by 8-base pair (bp) direct repeats of the H2B sequence. TU1 has long terminal inverted repeats approximately 840 bp long with an outer domain of 15-bp tandem repeats and a non-repeating inner domain, and is a member of a heterogeneous family of transposable elements. TU1 differs from most previously characterized eukaryotic transposable elements with terminal direct repats, but resembles the foldback transposon family in Drosophila.

Animals↗
PubMedDate not supplied

Smooth muscle alpha-action is a transformation-sensitive marker for mouse NIH 3T3 and Rat-2 cells.

Heteroploid mouse NIH 3T3 fibroblasts and several rat fibroblast strains (Rat-1, Rat-2 and REF-52) are cell lines of special interest in the field of carcinogenesis because of their extensive use as normal cells in transformation assays for putative cancer-causing genes. Exposure of these cells to carcinogenic chemicals or oncogenic DNA produces anchorage-independent cells with retracted cytoplasms that lack actin cables. All human fibroblast strains, normal and transformed, synthesize two electrophoretic forms of actin (beta- and gamma-actin). In contrast, we discovered that early-passage mouse and rat strains synthesize abundant amounts of each of the three electrophoretic forms of actin (alpha-, beta- and gamma-actin) but mouse and rat cancer cells express only beta- and gamma-actins. We now show that in NIH 3T3 and Rat-2 fibroblasts a third actin, the smooth muscle alpha isoform, is abundantly co-expressed with beta- and gamma-actin. In every instance tested following transformation to tumorigenicity, the accumulation of alpha-actin messenger RNA and alpha-actin synthesis was greatly inhibited. Shutdown of alpha-actin expression thus appears to be a reproducible transformation-sensitive marker in rodent fibroblasts.

Actins↗
PubMedDate not supplied

Altered expression of tropomodulin in cardiomyocytes disrupts the sarcomeric structure of myofibrils.

Tropomodulin is a tropomyosin-binding protein that terminates "pointed-end" actin filament polymerization. To test the hypothesis that regulation of tropomodulin:actin filament stoichiometry is critical for maintenance of actin filament length, tropomodulin levels were altered in cells by infection with recombinant adenoviral expression vectors, which produce either sense or antisense tropomodulin mRNA. Neonatal rat cardiomyocytes were infected, and sarcomeric actin filament organization was examined. Confocal microscopy indicated that overexpression of tropomodulin protein shortened actin filaments and caused myofibril degeneration. In contrast, decreased tropomodulin content resulted in the formation of abnormally long actin filament bundles. Despite changes in myofibril structure caused by altered tropomodulin expression, total protein turnover of the cardiomyocytes was unaffected. Biochemical analyses of infected cardiomyocytes indicated that changes in actin distribution, rather than altered actin content, accounted for myofibril reorganization. Ultrastructural analysis showed thin-filament disarray and revealed the presence of leptomeres after tropomodulin overexpression. Tropomodulin-mediated effects constitute a novel mechanism to control actin filaments, and our findings demonstrate that regulated tropomodulin expression is necessary to maintain stabilized actin filament structures in cardiac muscle cells.

Actins↗