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Biomedical subjects

D J Galas

Publications and source records attributed to D J Galas.

At least 19 recordsLinked to original sources
PubMed2001

Dynamic models of gene expression and classification.

Powerful new methods, like expression profiles using cDNA arrays, have been used to monitor changes in gene expression levels as a result of a variety of metabolic, xenobiotic or pathogenic challenges. This potentially vast quantity of data enables, in principle, the dissection of the complex genetic networks that control the patterns and rhythms of gene expression in the cell. Here we present a general approach to developing dynamic models for analyzing time series of whole genome expression. In this approach, a self-consistent calculation is performed that involves both linear and non-linear response terms for interrelating gene expression levels. This calculation uses singular value decomposition (SVD) not as a statistical tool but as a means of inverting noisy and near-singular matrices. The linear transition matrix that is determined from this calculation can be used to calculate the underlying network reflected in the data. This suggests a direct method of classifying genes according to their place in the resulting network. In addition to providing a means to model such a large multivariate system this approach can be used to reduce the dimensionality of the problem in a rational and consistent way, and suppress the strong noise amplification effects often encountered with expression profile data. Non-linear and higher-order Markov behavior of the network are also determined in this self-consistent method. In data sets from yeast, we calculate the Markov matrix and the gene classes based on the linear-Markov network. These results compare favorably with previously used methods like cluster analysis. Our dynamic method appears to give a broad and general framework for data analysis and modeling of gene expression arrays.

Animals↗
PubMed2000

DNA bending and twisting properties of integration host factor determined by DNA cyclization.

The binding of many proteins to DNA is profoundly affected by DNA bending, twisting, and supercoiling. When protein binding alters DNA conformation, interaction between inherent and induced DNA conformation can affect protein binding affinity and specificity. Integration host factor (IHF), a sequence-specific, DNA-binding protein of Escherichia coli, strongly bends the DNA upon binding. To assess the influence of inherent DNA bending on IHF binding, we took advantage of the high degree of natural static curvature associated with an IHF site on a 163-bp minicircle and measured the binding affinity of IHF for its recognition site contained on this DNA in both circular and linear form. IHF showed a higher affinity for the circular form of the DNA when compared to the linear form. In addition, the presence of IHF during DNA cyclization changed the topology of cyclization products and their ability to bind IHF, consistent with IHF untwisting DNA. These results show that inherent DNA conformation anisotropy is an important determinant of IHF binding affinity and suggests a mechanism for modulation of IHF activity by local DNA conformation.

Bacterial Proteins↗
PubMed1994

Interaction of Fis protein with DNA: bending and specificity of binding.

The Escherichia coli Fis protein is a dimeric DNA-binding protein whose specific binding sites share a weak consensus sequence. Use of the gel retardation technique indicates that binding of Fis on a linear DNA fragment leads to the formation of a ladder of defined retarded complexes, independently of the presence of a specific site. This non-specific binding of Fis is consistent with a model where equivalent low-affinity sites on a given fragment would be bound randomly and independently of each other by consecutive Fis dimers. Evidence is presented that non-specific binding of Fis can, however, induce an apparent site-specific conformational change in the DNA. This observation is discussed in terms of a model in which each Fis:DNA complex detected in gel retardation experiments actually represents a dynamic equilibrium of a fixed number of Fis dimers distributed on the fragment.

Carrier Proteins↗
PubMed1993

Important unanswered questions concerning radiation risk estimates.

While the most important radiobiological questions at present can be stated simply, arriving at the answers requires the development of a broad base of understanding to which collaborative interdisciplinary efforts that use a variety of techniques will make important contributions. In spite of the range of questions that remain unanswered, we probably know more about the effects of radiation than any other environmental agent. A sound and well-planned research program into the mechanisms of radiation carcinogenesis can help in many predictable ways. It will provide data needed for setting protection standards, of course, but it will also play a vital role in solving some of the enigmas of the mechanisms of carcinogenesis. In addition, the information gained by the approach focused on basic science will ultimately provide much better methods for extrapolating from cellular and animal studies to health effects in humans and thus for developing sound, science-based radiation risk estimates.

Animals↗
PubMed1990

Functional organization of the ends of IS1: specific binding site for an IS 1-encoded protein.

The IS 1-encoded protein InsA binds specifically to both ends of IS1, and acts as a repressor of IS1 gene expression and may be a direct inhibitor of the transposition process. We show here, using DNasel 'foot-printing' and gel retardation, that the InsA binding sites are located within the 24/25 bp minimal active ends of IS1 and that InsA induces DNA bending upon binding. Conformational modification of the ends of IS1 as a result of binding of the host protein integration host factor (IHF) to its site within the minimal ends has been previously observed. Using a collection of synthetic mutant ends we have mapped some of the nucleotide sequence requirements for InsA binding and for transposition activity. We show that sequences necessary for InsA binding are also essential for transposition activity. We demonstrate that InsA and IHF binding sites overlap since some sequence determinants are shared by both InsA and IHF. The data suggest that these ends contain two functional domains: one for binding of InsA and IHF, and the other for transposition activity. A third region, when present, may enhance transposition activity with an intact right end. This 'architecture' of the ends of IS1 is remarkably similar to that of IS elements IS10, IS50 and IS903.

Bacterial Proteins↗
PubMed1989

A simple method for site-directed mutagenesis using the polymerase chain reaction.

We have developed a general and simple method for directing specific sequence changes in a plasmid using primed amplification by the polymerase chain reaction (PCR). The method is based on the amplification of the entire plasmid using primers that include the desired changes. The method is rapid, simple in its execution, and requires only minute amounts of plasmid template DNA. It is significant that there are no special requirements for appropriately placed restriction sites in the sequence to be manipulated. In our system the yield of transformants was high and the fraction of them harboring plasmids with only the desired change was consistently about 80%. The generality of the method should make it useful for the direct alteration of most cloned genes. The only limitation may be the total length of the plasmid to be manipulated. During the study we found that the Taq DNA polymerase used for PCR adds on a single extra base (usually an A) at the end of a large fraction of the newly synthesized chains. These had to be removed by the Klenow fragment of DNA polymerase to insure restoration of the gene sequence.

Base Sequence↗
PubMed1988

Mutational analysis of the open reading frames in the transposable element IS1.

IS1 is one of the smallest transposable elements found in bacteria (768 bp). It contains eight overlapping open-reading-frames (ORFs) greater than 50 codons, designated insA to insG and insB'. To determine which of the ORFs actually code for proteins involved in transposition, we have introduced amber codons into each ORF by site-directed mutagenesis which make neutral changes in the overlapping ORFs. Each mutant IS1 was then tested for its ability to mediate cointegrate formation in Su+ and Su- backgrounds. The mutant elements were also tested for trans-complementation in an IS1-free Salmonella background. Our results show that the products of the insA and insB genes are the only ones essential for cointegrate formation. We suggest that other ORFs may, however, encode accessory proteins.

Amino Acid Sequence↗
PubMed1987

Escherichia coli integration host factor binds specifically to the ends of the insertion sequence IS1 and to its major insertion hot-spot in pBR322.

We report here that the ends of IS1 are bound and protected in vitro by the heterodimeric protein integration host factor (IHF). Under identical conditions, RNA polymerase binds to one of these ends (IRL) and protects a region that includes the sequences protected by IHF. Other potential sites within IS1, identified by their homology to the apparent consensus sequence, are not protected. Footprinting analysis of deletion derivatives of the ends demonstrates a correspondence between the ability of the end sequence to bind IHF and its ability to function as an end in transposition. Nonetheless, some transposition occurs in IHF- cells, indicating that IHF is not an essential component of the transposition apparatus. IHF also binds and protects four closely spaced regions within the major hot-spot for insertion of IS1 in the plasmid pBR322. This striking correlation of hot-spot and IHF-binding sites suggests a possible role for IHF in IS1 insertion specificity.

Bacterial Proteins↗
PubMed1987

Escherichia coli integration host factor bends the DNA at the ends of IS1 and in an insertion hotspot with multiple IHF binding sites.

The integration host factor of Escherichia coli (IHF) is a small, histone-like protein which participates in the integration of bacteriophage lambda into the E. coli chromosome and in a number of regulatory processes. Our recent footprinting analysis has shown that IHF binds specifically to the ends of the transposable element IS1, as well as to several sites within a short segment of the plasmid pBR322. We have extended our studies of the binding of the IHF molecule to these sites in vitro using a gel retardation assay. We report here that IHF bends the DNA upon binding, as judged from the strong cyclic dependence of the protein-induced mobility shift on the position of the binding site. Using cloned, synthetic ends of IS1 as substrates, we have found that some mutations within the conserved bases of the IHF consensus binding sequence abolish binding, and that alterations of the flanking sequences can greatly reduce IHF binding. The presence of multiple IHF sites on a single DNA fragment increases binding very little, indicating that IHF does not bind cooperatively in this complex. We discuss the possibility that DNA bending is related to the role IHF plays in forming and stabilizing nucleoprotein complexes, and suggest that bending at the IHF sites may be important to its diverse effects in the cell.

Bacterial Proteins↗
PubMed1987

Expression of proteins essential for IS1 transposition: specific binding of InsA to the ends of IS1.

The insertion sequence IS1 displays a complex array of open reading frames (ORF). In an attempt to identify those which encode polypeptide products, we have systematically placed each ORF under the control of the P1 promoter of phage lambda. In the expression system we used, only the product of the insA gene was present in high enough amounts to be detected by polyacrylamide gel electrophoresis. The production of InsA was further increased in a first codon hook-up to phage T7 transcriptional and translational initiation signals. Cell extracts from InsA overproducers display a DNA binding activity specific for the ends of IS1. This activity was identified as the InsA protein itself.

Amino Acid Sequence↗
PubMed1987

Artificial transposable elements in the study of the ends of IS1.

We have constructed artificial IS1-based transposons by attaching synthetic oligodeoxynucleotides, corresponding to the sequence of the ends of IS1, to a selectable DNA segment ['omega' fragment; Prentki and Krisch, Gene 29 (1984) 303-313]. These transposons were used to examine the sequence requirements at the ends for IS1 transposition. We show here that a 24- to 28-bp sequence from the left or right ends of IS1 is capable of transposition when present at both ends of the omega fragment in the correct orientation. Transposition activity requires the presence of an intact IS1 in cis on the same plasmid molecule. In trans, however, neither resident genomic copies of IS1, nor copies carried by a compatible, high-copy-number plasmid present in the same cell, complement the artificial transposons efficiently. Transposition frequencies in the presence of a cis-complementing IS1 are, however, similar to those of the naturally occurring IS1-based transposon, Tn9. In addition, transposition results in a 9-bp duplication in the target DNA molecule as is usually the case for insertion of the intact IS1. Using this system, we have obtained evidence indicating that the activity of a synthetic IS1 end is not determined exclusively by its sequence, but can be strongly enhanced by a second, wild-type end used in the transposition event. The data also show that single base pair mutations can exhibit a cumulative effect in reducing transposition activity.

Base Sequence↗
PubMed1986

Functional promoters created by the insertion of transposable element IS1.

We have isolated several insertions of the transposable element IS1 into the proximal promoter (P3) of the beta-lactamase gene of plasmid pBR322, which do not abolish resistance to ampicillin. Using a transcription termination module (omega), we have shown that the gene can be expressed from hybrid promoters, created by the insertion of IS1. The terminal inverted repeats of IS1 carry sequences partially homologous to the "-35" consensus region. Splicing either of these sequences to the existing "-10" region of the beta-lactamase promoter by transposition of IS1 at the proper distance results in the formation of an active hybrid promoter. This interpretation was confirmed by transcription studies in vitro. Gene expression from the hybrid promoters was found to be less efficient than from P3. However, the orientation of IS1 that contributes a "-35" with the greater homology to the known "-35" consensus sequence is significantly more efficient than the other. In addition, we were able to assign a strong determinant of IS1 polarity to a 254 base-pair internal segment of IS1. An examination of the ends of many insertion sequences leads us to expect that the phenomenon described here may occur with several of these transposable elements, and may have an unexpected evolutionary significance.

Base Sequence↗
PubMed1985

Rigorous pattern-recognition methods for DNA sequences. Analysis of promoter sequences from Escherichia coli.

The basic nature of the sequence features that define a promoter sequence for Escherichia coli RNA polymerase have been established by a variety of biochemical and genetic methods. We have developed rigorous analytical methods for finding unknown patterns that occur imperfectly in a set of several sequences, and have used them to examine a set of bacterial promoters. The algorithm easily discovers the "consensus" sequences for the -10 and -35 regions, which are essentially identical to the results of previous analyses, but requires no prior assumptions about the common patterns. By explicitly specifying the nature of the search for consensus sequences, we give a rigorous definition to this concept that should be widely applicable. We also have provided estimates for the statistical significance of common patterns discovered in sets of sequences. In addition to providing a rigorous basis for defining known consensus regions, we have found additional features in these promoters that may have functional significance. These added features were located on either side of the -35 region. The pattern 5', or upstream, from the -35 region was found using the standard alphabet (A, G, C and T), but the pattern between the -10 and the -35 regions was detectable only in a sub-alphabet. Recent results relating DNA sequence to helix conformation suggest that the former (upstream) pattern may have a functional significance. Possible roles in promoter function are discussed in this light, and an observation of altered promoter function involving the upstream region is reported that appears to support the suggestion of function in at least one case.

Base Sequence↗