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A Collick

Publications and source records attributed to A Collick.

At least 19 recordsLinked to original sources
PubMed2004

Transgenic mice showing inflammation-inducible overexpression of granulocyte macrophage colony-stimulating factor.

We used the promoter of the human C-reactive protein (CRP) gene to drive inflammation-inducible overexpression of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) in transgenic mice. Transgenic mice carrying a CRP/GM-CSF fusion gene show a >150-fold increases in circulating levels of GM-CSF within 6 h of intraperitoneal inoculation with 25 microg of lipopolysaccharide. However, some of the transgenic mice also display relatively high basal levels of GM-CSF in the absence of any obvious inflammatory stimulus. Raised basal levels of GM-CSF are associated with a number of pathological changes, including enlarged and histologically abnormal livers and spleens and with increases in the number and activation state of macrophages and granulocytes in the peripheral blood. Despite problems associated with the expression of such a potent pleiotropic cytokine as GM-CSF, the principle of inflammation-inducible expression of chimeric constructs has been shown to be feasible. Inducible expression systems such as that described here could be of potential use in the study of the role of cytokines in health and disease and in the development of disease-resistant strains of livestock.

Amino Acid Sequence↗
PubMed2000

Somatic versus germline mutation processes at minisatellite CEB1 (D2S90) in humans and transgenic mice.

The most variable human minisatellites show extreme germline instability dominated by complex intra-allelic rearrangements plus a lower frequency of inter-allelic transfers of repeat units. In contrast, little is known about somatic instability at such loci. We have therefore used single-molecule PCR to analyze mutation at minisatellite CEB1 (D2S90) in human blood DNA. Somatic mutants were rare and involved only relatively simple intra-allelic events, with no bias toward expansions, in sharp contrast to the complex gain-biased rearrangements seen in sperm. Somatic and germline mutation processes were further analyzed in mice transgenic for a cosmid insert containing CEB1. Mutant molecules in transgenic sperm and blood were detected but only at the low frequencies seen in human blood and arose mainly by simple duplications and deletions as seen for somatic mutations in human. These data suggest distinct pathways for germline and somatic CEB1 mutations with germline instability involving recombination-based repair of meiotic double-strand breaks and somatic mutation arising by replication slippage or mitotic recombination. The problem of transferring germline-specific features of minisatellite instability from human to mouse suggests, with other recent observations, that long-range chromatin conformation may be required for the recombination-based mode of germline instability at human minisatellites.

Alleles↗
PubMed1998

Current and future contributions of transgenic mice to the analysis of germline toxicology.

Evermore sophisticated tests are need to study germline toxicology. The gene conversion-based systems developed in Leicester and in the USA are steps in the right direction, but a lot of validation both in vivo and in vitro is required. Transgenic technology can also be used to research the biology of testis, so that we know more how to make it more human-like. If you talk to toxicologists, they always complain: 'but it 's only a rat, it's only a mouse, it's not a man'. In future, once we understand more biology--it might be possible to make the toxicological response of a transgenic mouse more human-like. As we all know, the testis is a complex biological system and it is only when we get a better understanding of what is going on to the fundamental level are such developments possible. Indeed, it might be possible to do even more exciting things, such as taking mitotic human tissue culture cells and to inducing them to enter meiosis in vitro. Such a system would be a natural complement to the in vitro tests widely used in industry.

Animals↗
PubMed1997

Human minisatellite MS32 (D1S8) displays somatic but not germline instability in transgenic mice.

Human minisatellite MS32 (D1S8) shows instability both in the germline and, at much lower levels, in somatic DNA. To investigate factors that influence somatic and germline mutation, large cosmid-based constructs containing MS32 were introduced into mice, bred to homozygosity and tested for instability in blood and sperm. Analysis of single copy and multicopy transgenic lines revealed somatic mutants occurring at a frequency comparable with that seen in man. As in humans, these mutants arose mainly by simple intra-allelic duplications and deletions. In contrast, analysis of sperm DNA from four different transgenic lines showed no trace of the complex recombination-based germline instability seen in man, even using PCR-based approaches capable of detecting very rare mutants. These data provide further evidence that germline and somatic mutation at human minisatellite MS32 occur via distinct pathways, that a major barrier exists to the transfer of germline instability from humans to mice and that the mouse germline appears to be protected from mitotic instability of the type seen in blood.

Alleles↗
PubMed1996

Instability of long inverted repeats within mouse transgenes.

Various sequences in the mammalian genomes are unstable. One class of sequence arrangement is long inverted repeats, which are known to be unstable in bacteria and yeast. While in mammals some evidence suggests that short inverted repeats (<10 bp long) may show instability, nothing is known about the stability of long inverted repeats. Here we describe two unrelated multicopy transgenes in the mouse (loci 109 and OX1-5), each of which contains a long inverted repeat that shows substantial mitotic instability. This instability also occurs in the germline so that mutant transgenes appear within pedigrees at a high frequency. The mutation processes acting at these two inverted repeats are complex and can involve insertion or deletion, and can result in stabilization of the transgene. At transgene 109 mutational events range from very small rearrangements at the centre of the inverted repeat to complete transgene deletion. In addition we show that the rates of mutation at the inverted repeat of transgene OX1-5 can vary between the male and female germlines and between inbred strains of mice, suggesting the possibility of a genetic analysis to identify loci that modulate inverted repeat instability.

Animals↗
PubMed1995

Mutation processes at human minisatellites.

Minisatellites provide one of the most experimentally tractable systems for studying tandem repeat instability in man. Analysis of mutation processes has been greatly aided by the development of single molecule methods for recovering de novo mutants, and of techniques for exploring allele structure in detail. Application of these approaches to man has shown that minisatellites do not primarily mutate by processes such as replication slippage and unequal crossover intrinsic to the tandem repeat array. Instead, germline repeat instability is largely regulated by cis-acting elements near the array and involves unexpectedly complex processes of gene conversion, of potential relevance to the biology of meiosis. These processes can be explored both in humans and, in principle, in transgenic mouse models of human repeat instability.

Alleles↗
PubMed1994

Variable germline and embryonic instability of the human minisatellite MS32 (D1S8) in transgenic mice.

Tandem repeat loci such as minisatellites and trinucleotide repeats frequently show instability. We have investigated mutation at human minisatellite MS32 (locus D1S8) transferred to transgenic mice. Three lines of hemizygous transgenic mice were studied. A single-copy line (110D) was seen to be relatively stable, whilst two multicopy lines showed structural instability of the transgene in pedigrees (lines 109 and 110A). For both these lines, mutant structures were detected as a result of mutation events having occurred in the germline or early embryo. Structural changes seen included gain or loss of minisatellite repeat units (110A and 109), alteration of DNA flanking the minisatellite repeat array (109 only) or deletion of the entire transgene (109 only). This work demonstrates that tandem repeat transgenes can show instability and thus provide additional systems for the analysis of repetitive DNA structural change in mice.

Animals↗
PubMed1994

Tandemly repeated transgenes of the human minisatellite MS32 (D1S8), with novel mouse gamma satellite integration.

The human hypervariable minisatellite MS32 has a well characterised internal repeat unit array and high mutation rates have been observed at this locus. Analysis of MS32 mutants has shown that male germline mutations are polarised to one end of the array and frequently involve complex gene conversion-like events, suggesting that tandem repeat instability may be modulated by cis-acting sequences flanking the locus. In order to investigate the processes affecting MS32 mutation rate and mechanism, we have created transgenic mice harbouring an MS32 allele. Here we describe the organisation of eight transgenic insertions. Analysis of these transgenic loci by MVR-PCR and structural analysis of the junctions between mouse flanking DNA and the transgenic loci has shed light on mechanisms of integration and rearrangement of the tandem repeated transgenes. Sequence analysis of the mouse DNA flanking these transgenes has shown that 5 of the 8 insertions have integrated into mouse gamma satellite repeated sequence. This suggests a non-random integration of the MS32 transgene construct into the mouse genome.

Alleles↗
PubMed1994

Use of vectorette and subvectorette PCR to isolate transgene flanking DNA.

Vectorette PCR permits the specific amplification of DNA segments flanking a known DNA sequence. It enables the application of the PCR where sequence information is only available for one primer site. We now show that vectorette PCR can be used for the systematic mapping and retrieval of transgene flanking DNA. We also show that the sequence of large vectorette PCR fragments can be obtained without cloning, by the production of subvectorette fragments.

Animals↗
PubMed1993

A tetranucleotide repeat mouse minisatellite displaying substantial somatic instability during early preimplantation development.

The highly variable mouse minisatellite Hm-2 is located on chromosome 9 and consists of GGCA tetranucleotide repeats with alleles containing up to 5000 repeat units. This locus is unstable with a germline mutation rate to new length alleles of at least 3.6% per gamete. Hm-2 also shows substantial somatic instability, producing mutational mosaicism detectable in 20% of adult mice. Analysis of allele dosage in mice carrying somatic mutations, plus studies of mosaicism in mouse embryos and extraembryonic tissues, suggests that somatic mutant alleles preferentially arise during preimplantation development and particularly during the first two cell divisions after fertilization.

Alleles↗
PubMed1993

Minisatellite variant repeat mapping: application to DNA typing and mutation analysis.

Most DNA typing systems assay allele length variation at tandemly repeated loci such as minisatellites and microsatellites. Allele length measurements are approximate, which impedes the use of such loci in forensic analysis and in studies of allelic variability at hypervariable loci. We now review progress in the development of alternative DNA typing systems based on allelic variation in the interspersion patterns of variant repeat units along minisatellite alleles. Minisatellite variant repeat mapping by PCR (MVR-PCR) not only provides a powerful new digital approach to DNA typing, but also for the first time allows investigation of the true level of allelic variability at minisatellite loci and of the mutational mechanisms that generate ultravariability.

Alleles↗
PubMed1991

Minisatellite binding protein Msbp-1 is a sequence-specific single-stranded DNA-binding protein.

Msbp-1 is a minisatellite-specific DNA-binding protein. Using synthetic binding substrates, we now show that Msbp-1 binds not to double-stranded DNA, but exclusively to single-stranded DNA. Binding is specific to the guanine-rich strand of the minisatellite duplex, interactions with the cytosine-rich strand being undetectable by southwestern analysis. Furthermore, the binding site required for successful DNA-protein interactions appears to be two or more minisatellite repeat units. We have also isolated, by whole-genome PCR and cloning, one Msbp-1 binding site from the human genome. Again, the binding strand of this molecule contains a repetitive G-rich structure equivalent to that of a small minisatellite. These observations are discussed with respect to other single-stranded DNA-binding proteins known to play a role in recombination processes.

Animals↗
PubMed1991

Spontaneous mutation at the hypervariable mouse minisatellite locus Ms6-hm: flanking DNA sequence and analysis of germline and early somatic mutation events.

Hypervariability at minisatellite loci is maintained by spontaneous mutation to new-length alleles. At the most variable loci, mutation rate is directly measurable by pedigree analysis. The mouse minisatellite locus Ms6-hm has a germline mutation rate of 2.5% per gamete and is therefore one of the most unstable loci yet identified in the mouse genome. Mutation events at this locus also occur during early mouse development, resulting in mice mosaic for cells carrying a common non-parental allele in different somatic tissues and the germline. The DNA sequence flanking Ms6-hm is rich in dispersed repetitive elements; the minisatellite array has expanded from within a member of the Mouse Transcript family which is flanked by two additional Mouse Transcript elements, and a B2 element lies further 3' to the minisatellite. To define the characteristics of mutation events at Ms6-hm we have analysed 19 germline and 13 somatic length-change events. Germline mutation events at Ms6-hm are not accompanied by the exchange of flanking markers in three informative mutant alleles analysed.

Alleles↗
PubMed1991

Principles and recent advances in human DNA fingerprinting.

Since 1985, DNA typing systems have played an increasingly important role in many aspects of human genetics, most notably in forensic and legal medicine. This article reviews the development of multilocus and single locus minisatellite DNA probes, and more recently the use of PCR to amplify hypervariable DNA loci, as well as discussing the biological properties of the unstable regions of DNA which form the basis of almost all DNA fingerprinting systems.

Base Sequence↗
PubMed1990

Detection of a novel minisatellite-specific DNA-binding protein.

We describe the detection of a ubiquitous DNA-binding protein which appears to interact specifically with tandem-repeated minisatellites. The murine 40 kd protein, which we term Msbp-1, was found to be present in all mouse tissues tested. This protein was bound specifically and with high affinity by double-stranded DNA containing a repeat sequence related to the minisatellite 'core' sequence, and binding required the presence of multiple repeat units. Corresponding minisatellite-specific DNA-binding proteins could also be detected in species ranging from Drosophila to man. This analysis represents the first direct evidence that minisatellites can function as a specific recognition signal for an endogenous DNA-binding protein.

Animals↗
PubMed1989

Characterization of a highly unstable mouse minisatellite locus: evidence for somatic mutation during early development.

A highly unstable mouse minisatellite locus, Ms6-hm, has been identified in mouse DNA fingerprints produced by cross-hybridization with human minisatellite probe 33.6. A 7-kb allele of Ms6-hm was cloned from a C57BL/6J mouse and collapsed to a 400-bp plasmid insert on propagation in Escherichia coli due to loss of the majority of minisatellite repeat units. Sequence analysis revealed that Ms6-hm has evolved by amplification within a member of the MT (mouse transcript) family of interspersed repetitive elements. Linkage analysis localized Ms6-hm near the brown coat color gene (b) on chromosome 4. Multiallelism and heterozygosity at this locus within inbred strains result from a high germline mutation rate to new-length alleles (2.5% per gamete). Mice mosaic for cells carrying a nonparental allele in somatic tissue, and in some cases also in the germline, provide evidence for additional, somatic, mutation events at Ms6-hm. In two mosaic mice the fraction of cells containing the nonparental allele has been shown to be indistinguishable in different adult tissues. These somatic mutation events at Ms6-hm must therefore occur very early in development, preceding the allocation of somatic lineages, and the same pool of primitive ectoderm cells must contribute equally to all somatic tissues. Under low-stringency hybridization conditions the collapsed subclone of Ms6-hm cross-hybridizes to other unstable loci in the mouse genome to generate a novel and highly individual specific mouse DNA fingerprint.

Alleles↗
PubMed1988

CpG methylation of an X-linked transgene is determined by somatic events postfertilization and not germline imprinting.

The process of X-inactivation in mammals requires at least two events, the initiation of inactivation and the maintenance of the inactive state. One possible mechanism of control is by methylation of DNA at CpG dinucleotides to maintain the inactive state. Furthermore, the paternal X-chromosome is frequently inactivated in the extraembryonic membranes. The relationship between the parental origin of the chromosome, nonrandom inactivation and DNA methylation is not clear. In this paper, we report on the CpG methylation of an X-linked transgene, CAT-32. The levels of methylation in embryonic, extraembryonic and germline cells indicates that the modifications of the transgene are broadly similar to those reported for endogenous X-linked genes. Interestingly, the methylation of CAT-32 transgene in extraembryonic tissues displays patterns that could be linked to the germline origin of each allele. Hence, the maternally derived copy of CAT-32 was relatively undermethylated when compared to the paternal one. The changes in DNA methylation were attributed to de novo methylation occurring after fertilization, most probably during differentiation of extraembryonic tissues. In order to determine whether or not the patterns of DNA methylation reflected the germline origin of the X-chromosome, we constructed triploid embryos specifically to introduce two maternal X-chromosomes in the same embryo. In some of these triploid conceptuses, methylation patterns characteristic of the paternally derived transgene were observed. This observation indicates that the methylation patterns are not necessarily dependent on the parental origin of the X-chromosome, but could be changed by somatic events after fertilization. One of the more likely mechanisms is methylation of the transgene following inactivation of the X-chromosome in extraembryonic tissues.

Alleles↗