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R Lauster

Publications and source records attributed to R Lauster.

33 records · Page 2Linked to original sources
PubMed1996

Functional significance of polymorphism among MHC class II gene promoters.

The functional significance of polymorphism among MHC class II promoters in man and mouse is here reviewed, mainly in terms of the hypothesis of differential expression. The hypothesis proposes that differences between antigen-presenting cells in MHC class II expression exert a co-dominant effect on the Th1-Th2 cytokine balance, such that class II molecules of one type come to control to a greater extent the production of one group of cytokines, and those of another type the production of the alternative group. The survey deals with the influence of signal strength and antigen-presenting cell type on T-cell subset differentiation; functional differences between MHC class II molecules not obviously related to determinant selection; disease protection mediated by HLA alleles; mechanisms possibly responsible for allotypic and isotypic bias; overdominance (heterozygous advantage) in selection for expression of class II alleles; MHC class II promoter structure and function; inter-locus and inter-allele variability within human MHC class II gene upstream regulatory regions; a comparison of these polymorphisms in mouse and man; read-out of class II promoter function; and a comparison with expression of MHC class I. We conclude that the evidence that this variation is functionally active (i.e. controls expression) is increasing, but is not yet compelling. The crucial test still to come, we suggest, is whether or not the biological effects attributable to this polymorphism will line up with molecular studies on expression.

Alleles↗
PubMed1995

The chicken immunoglobulin lambda light chain gene is transcriptionally controlled by a modularly organized enhancer and an octamer-dependent silencer.

Characterization of the regulatory elements involved in V(D)J recombination is crucial for understanding development of the B and T cell immune repertoire. Previously we have shown that the chicken immunoglobulin lambda light chain gene (CLLCG) undergoes lymphoid-specific rearrangement in transgenic mice. The whole gene is only 10 kb in length and contains all phylogenetically conserved target sites for recombinational and transcriptional regulation. In this study we have localized an enhancer element in a region 4 kb downstream of the constant (C) region. The 467 bp element can be subdivided into three subfragments. The previously detected silencer element on the V-J intervening sequence is shown to be localized on a 500 bp fragment. Partial silencer activity is retained on a 250 bp fragment, which includes an octamer motif. By mutational analysis this octamer is shown to be essential for B cell- but not for T cell-specific silencer function. The silencer represses transcription directed by heterologous elements like the SV 40 promoter or the Ig kappa 3' enhancer. We propose that transcription of the unrearranged and rearranged Ig genes is regulated by complex interactions between different modules from the promoter, enhancer and silencer, which is eliminated by recombination during B cell development.

Animals↗
PubMed1994

Rearrangement of the chicken lambda light chain locus: a silencer/antisilencer regulation.

Chicken immunoglobulin lambda light chain rearrangement is regulated by different controlling elements. One negative control element, acting as a strong transcriptional silencer, is located in the V-J intervening sequence which is excised during the rearrangement process. Positive control elements include the V lambda promoter, the enhancer of transcription (3' of C lambda), and one (or two) putative antisilencer element(s) located on each side of the silencer. It is proposed that these antisilencer elements counteract transiently the silencer thus allowing rearrangement of one allele in chicken B cell progenitors. The implications of this regulation for mouse B cell development are discussed.

Animals↗
PubMed1993

Promoter, enhancer and silencer elements regulate rearrangement of an immunoglobulin transgene.

The chicken Ig lambda light chain locus is composed of a single V gene closely linked (1.8 kb) to a single J-C unit in its natural configuration. In mice transgenic for this locus, the transgene becomes rearranged in B cells and to a much lesser extent in T cells. Modifications were introduced in the transgene in order to characterize elements which target the recombinase to the Ig loci. In the absence of either the promoter or the enhancer located 3' of C lambda, rearrangement of the transgene is reduced 20- to 100-fold. Moreover, rearrangement is increased 5-fold when the DNA segment between V lambda and J lambda ('Uo segment'), which is deleted during the joining process, is replaced by a neutral DNA segment of equal length. The Uo segment behaved as a strong transcriptional silencer when tested in a CAT assay in vitro. Control transgenic mice harbouring only the two 3 bp mutations that introduced restriction sites at both ends of the Uo segment to allow for its replacement were also analysed. Rearrangement was reduced 10- to 100-fold in B cells from such transgenic lines. A model is proposed whereby the sites of these two mutations would function by counteracting transiently the repressing effect of the silencer, thus giving access of the chicken light chain locus to the recombinase.

Animals↗
PubMed1989

Cytosine-specific type II DNA methyltransferases. A conserved enzyme core with variable target-recognizing domains.

Comparisons of the amino acid sequences of m5C DNA methyltransferases (Mtases) from 11 prokaryotes and one eukaryote reveal a very similar organization. Among all the enzymes one can distinguish highly conserved "core" sequences and "variable" regions. The core sequences apparently mediate steps of the methylation reaction that are common to all the enzymes. The major variable region has been shown in our previous studies on multispecific phage Mtases to contain the target-recognizing domains (TRDs) of these enzymes. Here we have compared the amino acid sequences of various TRDs from phage Mtases. This has revealed the presence of both highly conserved and variable amino acids. We postulate that the conserved residues represent a "consensus" sequence defining a TRD, whereas the specificity of the TRD is determined by the variable residues. We have observed similarity between this consensus sequence and sequences in the variable region of the monospecific Mtases. We predict that the regions thus identified represent part of the TRDs of monospecific Mtases.

Amino Acid Sequence↗
PubMed1989

Evolution of type II DNA methyltransferases. A gene duplication model.

On the basis of consensus sequences, which had previously been defined for two groups of closely related cytosine-specific and adenine-specific DNA methyltransferases, homologies can be detected that indicate a common origin for these proteins. Intramolecular comparisons of several of these enzymes reveal homology relationships, which suggests that gene duplication is a phylogenetic principle in the evolution of the Mtases. One or two duplications of an ancestral gene encoding a 12,000 to 16,000 Mr protein, followed by divergent evolution, may have led to very different protein structures and could explain the differences in amino acid sequences, molecular weights and biochemical properties. Intermolecular and intramolecular homologies were also recognized in type II restriction endonucleases, suggesting a very similar evolutionary pathway.

Amino Acid Sequence↗
PubMed1988

Sequential order of target-recognizing domains in multispecific DNA-methyltransferases.

In the multispecific DNA(cytosine-5)-methyltransferases (Mtases) of Bacillus subtilis phages SPR and phi 3T the domains responsible for recognition of DNA methylation targets CCA/TGG, CCGG, GGCC (SPR) and GCNGC, GGCC (phi 3T) represent contiguous sequences of approximately 50 amino acids each. These domains are tandemly arranged and do not overlap. They are part of a 'variable' segment within the enzymes which is flanked by 'conserved' amino acids, which are very similar amongst bacterial monospecific and the multispecific Mtases studied here. These results follow from a mutational analysis of the SPR and phi 3T Mtase genes. They further support our concept of a modular enzyme organization, according to which variability of type II Mtases with respect to target recognition is achieved by a combination of the same enzyme core with a variety of target-recognizing domains.

Amino Acid Sequence↗
PubMed1987

The GATATC-modification enzyme EcoRV is closely related to the GATC-recognizing methyltransferases DpnII and dam from E. coli and phage T4.

The amino acid sequence of EcoRV DNA methyltransferase which methylates the amino group of the 5'-adenine residue of the target sequence GATATC has been found to be closely related to that of three other adenine methyltransferases, DpnII, dam and damT4, the target sequence of which is GATC. Despite large differences on the DNA level, the four sequences show four blocks of homologies. One of these blocks has the sequence DVYXDPPY and is found with little modification in numerous other DNA methyltransferases. It is speculated that it could be the binding site of the methyl donor, S-adenosylmethionine. On the other hand, the identification of a DNA-binding region is more tenuous. As expected, no analogies with (dimeric) repressors and cro proteins which have the characteristic helix-turn-helix motif have been observed.

Amino Acid Sequence↗
PubMed1987

Construction and use of chimeric SPR/phi 3T DNA methyltransferases in the definition of sequence recognizing enzyme regions.

Multispecific DNA methyltransferases (Mtases) of temperate Bacillus subtilis phages SPR and phi 3T methylate the internal cytosine of the sequence GGCC. They differ in their capacity to methylate additional sequences. These are CCGG and CC(A/T)GG in SPR and GCNGC in phi 3T. Introducing unique restriction sites at equivalent locations within the two genes facilitated the construction of chimeric genes. These expressed Mtase activity at a level comparable to that of the parental genes. The methylation specificity of chimeric enzymes was correlated with the location of chimeric fusions. This analysis, which also included the use of mutant genes, showed that domains involved in the recognition of target sequences unique to each enzyme [CCGG, CC(A/T)GG or GCNGC] are represented by the central non-conserved parts of the proteins, whilst recognition of the sequence (GGCC), which is a target for both enzymes, is determined by an adjacent conserved region.

Bacillus subtilis↗
PubMed1987

Organization of multispecific DNA methyltransferases encoded by temperate Bacillus subtilis phages.

B. subtilis phage rho 11s codes for a multispecific DNA methyltransferase (Mtase) which methylates cytosine within the sequences GGCC and GAGCTC. The Mtase gene of rho 11s was isolated and sequenced. It has 1509 bp, corresponding to 503 amino acids (aa). The enzyme's Mr of 57.2 kd predicted from the nucleotide sequence was verified by direct Mr determinations of the Mtase. A comparison of the aa sequence of the rho 11s Mtase with those of related phages SPR and phi 3%, which differ in their methylation potential, revealed generalities in the building plan of such enzymes. At least 70% of the aa of each enzyme are contained in two regions of 243 and 109 aa at the N and C termini respectively, which are highly conserved among the three enzymes. In each enzyme, variable sequences separate the conserved regions. Variability is generated through the single or multiple use of related and unrelated sequence motifs. We propose that the recognition of those DNA target sequences, which are unique for each of the three enzymes, is determined by these variable regions. Evolutionary relationships between the three enzymes are discussed.

Amino Acid Sequence↗
PubMed1986

Multispecific DNA methyltransferases from Bacillus subtilis phages. Properties of wild-type and various mutant enzymes with altered DNA affinity.

Temperate Bacillus subtilis phages SPR, phi 3T, rho 11 and SP beta code for DNA methyltransferases, each having multiple sequence specificities. The SPR wild-type and various mutant methyltransferases were overproduced 1000-fold in Escherichia coli and were purified by three consecutive chromatographic steps. The stable form of these multispecific enzymes in solution are monomers with a relative molecular mass (Mr) of about 50,000. The methyl-transfer kinetics of the SPR wild-type and mutant enzymes were determined with DNA substrates carrying either none or one of the three recognition sequences (GGCC, CCGG, CCATGG). Evaluation of the catalytic properties for DNA and S-adenosylmethionine binding suggested that the NH2-terminal part of the protein is important for both non-sequence-specific DNA binding and S-adenosylmethionine binding as well as transfer of methyl groups. On the other hand, mutations in the COOH-terminal part lead to weaker site-specific interactions of the enzyme. Antibodies raised against the purified SPR enzyme specifically immunoprecipitated the phi 3T, rho 11 and SP beta methyltransferases, bu failed to precipitate the chromosomally coded enzymes from B. subtilis (BsuRI) and B. sphaericus (BspRI). Immunoaffinity chromatography is an efficient purification step for the related phage methyltransferases.

Bacillus subtilis↗
PubMed1986

Cloning and expression of Bacillus subtilis phage DNA methyltransferase genes in Escherichia coli and B. subtilis.

The DNA methyltransferase (Mtase) genes of the temperate Bacillus subtilis phages SPR (wild type and various mutants), phi 3T, rho 11 and SP beta have been cloned and expressed in Escherichia coli and B. subtilis host-plasmid vector systems. Mtase activity has been quantitated in these clones by performing in vitro methylation assays of cell-free extracts. The four-phage Mtase genes differ in the amount of Mtase synthesized when transcribed from their genuine promoters. In B. subtilis as well as in E. coli the SPR Mtase is always produced in smaller amounts than the other phage Mtases. Expression levels of the SPR Mtase are dependent on the strength of the upstream vector promoter sequences. Overproduction of the SPR wild-type and mutant enzymes was achieved in E. coli (inducible expression) by fusions to the lambda pL or the tac promoter and in B. subtilis (constitutive expression) by means of the phage SP02 promoter.

Bacillus subtilis↗