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Biomedical subjects

D Galas

Publications and source records attributed to D Galas.

14 recordsLinked to original sources
PubMed2001

Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein.

Sclerosteosis is an autosomal recessive sclerosing bone dysplasia characterized by progressive skeletal overgrowth. The majority of affected individuals have been reported in the Afrikaner population of South Africa, where a high incidence of the disorder occurs as a result of a founder effect. Homozygosity mapping in Afrikaner families along with analysis of historical recombinants localized sclerosteosis to an interval of approximately 2 cM between the loci D17S1787 and D17S930 on chromosome 17q12-q21. Here we report two independent mutations in a novel gene, termed "SOST." Affected Afrikaners carry a nonsense mutation near the amino terminus of the encoded protein, whereas an unrelated affected person of Senegalese origin carries a splicing mutation within the single intron of the gene. The SOST gene encodes a protein that shares similarity with a class of cystine knot-containing factors including dan, cerberus, gremlin, prdc, and caronte. The specific and progressive effect on bone formation observed in individuals affected with sclerosteosis, along with the data presented in this study, together suggest that the SOST gene encodes an important new regulator of bone homeostasis.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse.

Scurfy (sf) is an X-linked recessive mouse mutant resulting in lethality in hemizygous males 16-25 days after birth, and is characterized by overproliferation of CD4+CD8- T lymphocytes, extensive multiorgan infiltration and elevation of numerous cytokines. Similar to animals that lack expression of either Ctla-4 or Tgf-beta, the pathology observed in sf mice seems to result from an inability to properly regulate CD4+CD8- T-cell activity. Here we identify the gene defective in sf mice by combining high-resolution genetic and physical mapping with large-scale sequence analysis. The protein encoded by this gene (designated Foxp3) is a new member of the forkhead/winged-helix family of transcriptional regulators and is highly conserved in humans. In sf mice, a frameshift mutation results in a product lacking the forkhead domain. Genetic complementation demonstrates that the protein product of Foxp3, scurfin, is essential for normal immune homeostasis.

Amino Acid Motifs↗
PubMed1996

A mathematical analysis of in vitro molecular selection-amplification.

We construct a mathematical model for in vitro molecular selection with amplification. Using DNA-protein binding as the illustrative example, we obtain an expression for the probability that a randomly selected molecule from the final in vitro selection products is a molecule with the highest binding affinity. Experiments of this type have been reported for several examples of DNA binding proteins. Our study requires a model of the DNA-protein binding constant between DNA molecules and the target protein. The relationship between binding constants and selection probabilities is presented under simplifying but reasonable assumptions. From our analysis, we find that for successful in vitro selection experiments there should be a certain relationship between the number of polymerase chain reaction cycles and the concentration of free protein. The results obtained should be widely applicable to a variety of selection-amplification procedures.

DNA↗
PubMed1991

Translational control of transposition activity of the bacterial insertion sequence IS1.

The experiments reported here provide strong evidence indicating that the transposition frequency of the bacterial insertion sequence IS1 is determined principally by two IS1-specified proteins. The first, InsA, was previously shown to bind to the ends of the element and to act as a repressor. We present both physical and genetic evidence which reveals that the second, the InsAB' transposase, is a fusion of InsA with the product of a downstream reading frame, InsB'. Synthesis of this protein occurs by a -1 frameshift between the insA and insB' frames. It requires the presence of an intact retroviral-like frameshift signal composed of an A6C motif and a downstream region able to form several alternative secondary structures. In vivo studies show that IS1 transposition activity depends on the relative rather than on the absolute levels of InsA and InsAB'. The ratio is determined primarily at the translational level by frameshifting and appears to be relatively insensitive to large variations in levels of transcription. This novel homeostatic control could therefore protect IS1 from activation as a consequence of insertion into active transcription units.

Base Composition↗
PubMed1990

Optimizing restriction fragment fingerprinting methods for ordering large genomic libraries.

We present a statistical analysis of the problem of ordering large genomic cloned libraries through overlap detection based on restriction fingerprinting. Such ordering projects involve a large investment of effort involving many repetitious experiments. Our primary purpose here is to provide methods of maximizing the efficiency of such efforts. To this end, we adopt a statistical approach that uses the likelihood ratio as a statistic to detect overlap. The main advantages of this approach are that (1) it allows the relatively straightforward incorporation of the observed statistical properties of the data; (2) it permits the efficiency of a particular experimental method for detecting overlap to be quantitatively defined so that alternative experimental designs may be compared and optimized; and (3) it yields a direct estimate of the probability that any two library members overlap. This estimate is a critical tool for the accurate, automatic assembly of overlapping sets of fragments into islands called "contigs." These contigs must subsequently be connected by other methods to provide an ordered set of overlapping fragments covering the entire genome.

Genomic Library↗
PubMed1990

The regulatory role of the IS1-encoded InsA protein in transposition.

We show here that the protein InsA, which is encoded by IS1 and binds specifically to the terminal inverted repeats of this insertion sequence, negatively regulates IS1 transposition activity. We demonstrate that it inhibits both IS1-mediated cointegrate formation and transposition of a synthetic IS1-based transposon ('omegon'; omega-on). These results also indicate that the omega-on which does not itself encode IS1 transposition functions can be complemented in trans, presumably by the copies of IS1 resident in the Escherichia coli chromosome. Using insA-lacZ gene fusions, we show that at least part of this effect can be explained by the ability of InsA to repress expression of IS1-encoded genes both in cis or in trans. The experiments involving omega-on transposition raise the possibility that InsA inhibits transposition directly by competition with the transposase for their cognate site within the ends of IS1.

Base Sequence↗
PubMed1989

Single-sperm typing: determination of genetic distance between the G gamma-globin and parathyroid hormone loci by using the polymerase chain reaction and allele-specific oligomers.

The frequency of recombination between the G gamma-globin (HBG2) and parathyroid hormone (PTH) loci on the short arm of human chromosome 11 was estimated by typing greater than 700 single-sperm samples from two males. The sperm-typing technique employed involves the polymerase chain reaction and allele-specific oligonucleotide hybridization. Our maximum likelihood recombination fraction estimate of 0.16 (95%) confidence interval, 0.13-0.19) falls well within previous estimates based on family studies. With current technology and a sample size of 1000 sperm, recombination fractions down to approximately 0.009 can be estimated with statistical reliability; with a sample size of 5000 sperm, this value drops to about 0.004. Reasonable technological improvements could result in the detection of recombination frequencies less than 0.001.

Alleles↗
PubMed1986

Replication of pSC101: effects of mutations in the E. coli DNA binding protein IHF.

We have shown that the plasmid pSC101 is unable to be maintained in strains of E. coli carrying deletions in the genes himA and hip which specify the pleitropic heterodimeric DNA binding protein, IHF. We show that this effect is not due to a modulation of the expression of the pSC101 RepA protein, required for replication of the plasmid. Inspection of the DNA sequence of the essential replication region of pSC101 reveals the presence of a site, located between the DnaA binding-site and that of RepA, which shows extensive homology with the consensus IHF binding site. The proximity of the sites suggests that these three proteins, IHF, DnaA, and RepA may interact in generating a specific DNA structure required for initiation of pSC101 replication.

Amino Acid Sequence↗
PubMed1985

Specificity of insertion of IS1.

A systematic study of the specificity of insertion of the transposable element IS1 into small defined-sequence plasmids (pBR322 and derivatives) was conducted to determine the features of the DNA sequence that influence target site selection. We have physically mapped several collections of independent insertions of IS1 into these plasmids and have determined: (1) that about 80% of all insertions occur in the DNA segment (about 200 base-pairs) between the unique EcoRI site of pBR322 and the beginning of the beta-lactamase gene, one of the two regions of high A + T density in this plasmid; (2) that there is a strong orientation effect in this region (almost all IS1 insertions are in one orientation) that depends on both the pBR322 sequence and the environment of the transposon in the donor molecule; and (3) that the orientation effect does not depend on the strong transcription that is directed through this region in pBR322. Furthermore, we have found that insertion of a poly(dA X dT) segment into pBR322 creates an artificial hotspot for IS1 insertion, even though it is not as attractive for insertion as the above-mentioned major hotspot. Our observations suggest that an interplay between several properties of the target sequences and the sequence environment of the donor transposon is responsible for the observed specificity of position and orientation. One of the possibilities discussed here is that preferred "entry-sites", or "signal" sequences, for the transposition complex play a major role in determining the positions and orientations of IS1 insertions.

Ampicillin↗
PubMedDate not supplied

DNA sequence at the end of IS1 required for transposition.

The insertion sequence IS1 belongs to a class of bacterial transposable genetic elements that can form compound transposons in which two copies of IS1 flank an otherwise non-transposable segment of DNA. IS1 differs from other known elements of this class (such as IS10, IS50 and IS903) in several respects. It is one of the smallest known insertion elements, exhibits a relatively complex array of open reading frames, is present in the chromosomes of various Enterobacteria, in some cases in many copies, and its insertion can result in the duplication of either 8 or 9 base pairs (bp) in the target DNA. Furthermore, although, like other members of the compound class, it seems to undergo direct transposition, IS1 also promotes replicon fusion (co-integrate formation) at a relatively high frequency. Like all other elements studied to date, the integrity of the extremities of IS1 are essential for efficient transposition. We have constructed a test system to determine the minimal DNA sequences at the extremities of IS1 required for transposition. Sequential deletions of the end sequences reveal that 21-25 bp of an isolated extremity are sufficient for transposition. A specific sequence 13-23 bp from the ends, defining the edge of the minimal sequence, is implicated as an essential site. The sites, symmetrically arrayed at both ends of IS1, correspond to the apparent consensus sequence of the known binding sites for the Escherichia coli DNA-binding protein (called integration host factor or IHF) which is required for the site-specific recombination that leads to integration of bacteriophage lambda into the bacterial genome. The sites at the ends of IS1 may thus bind a host protein, such as JHF or a related protein, that is involved in regulating the transposition apparatus.

Base Sequence↗