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M D McLean

Publications and source records attributed to M D McLean.

13 recordsLinked to original sources
PubMed2000

Alteration of cardiac myofibrillogenesis by liposome-mediated delivery of exogenous proteins and nucleic acids into whole embryonic hearts.

A precise organization of contractile proteins is essential for contraction of heart muscle. Without a necessary stoichiometry of proteins, beating is not possible. Disruption of this organization can be seen in diseases such as familial hypertrophic cardiomyopathy and also in acquired diseases. In addition, isoform diversity may affect contractile properties in such functional adaptations as cardiac hypertrophy. The Mexican axolotl provides an uncommon model in which to examine specific proteins involved with myofibril formation in the heart. Cardiac mutant embryos lack organized myofibrils and have altered expression of contractile proteins. In order to replicate the disruption of myofibril formation seen in mutant hearts, we have developed procedures for the introduction of contractile protein antibodies into normal hearts. Oligonucleotides specific to axolotl tropomyosin isoforms (ATmC-1 and ATmC-3), were also successfully introduced into the normal hearts. The antisense ATmC-3 oligonucleotide disrupted myofibril formation and beating, while the sense strands did not. A fluorescein-tagged sense oligonucleotide clearly showed that the oligonucleotide is introduced within the cells of the intact hearts. In contrast, ATmC-1 anti-sense oligonucleotide did not cause a disruption of the myofibrillar organization. Specifically, tropomyosin expression can be disrupted in normal hearts with a lack of organized myofibrils. In a broader approach, these procedures for whole hearts are important for studying myofibril formation in normal hearts at the DNA, RNA, and/or protein levels and can complement the studies of the cardiac mutant phenotype. All of these tools taken together present a powerful approach to the elucidation of myofibrillogenesis and show that embryonic heart cells can incorporate a wide variety of molecules with cationic liposomes.

Ambystoma mexicanum↗
PubMed1999

Expression of axolotl RNA-binding protein during development of the Mexican axolotl.

Amphibians occupy a central position in phylogeny between aquatic and terrestrial vertebrates and are widely used as model systems for studying vertebrate development. We have undertaken a comprehensive molecular approach to understand the early events related to embryonic development in the Mexican axolotl, Ambystoma mexicanum, which is an exquisite animal model for such explorations. Axolotl RBP is a RNA-binding protein which was isolated from the embryonic Mexican axolotl by subtraction hybridization and was found to show highest similarity with human, mouse, and Xenopus cold-inducible RNA-binding protein (CIRP). The reverse transcriptase polymerase chain reaction (RT-PCR) analysis suggests that it is expressed in most of the axolotl tissues except liver; the expression level appears to be highest in adult brain. We have also determined the temporal and spatial pattern of its expression at various stages of development. RT-PCR and in situ hybridization analyses indicate that expression of the AxRBP gene starts at stage 10-12 (gastrula), reaches a maxima around stage 15-20 (early tailbud), and then gradually declines through stage 40 (hatching). In situ hybridization suggests that the expression is at a maximum in neural plate and neural fold at stage 15 (neurula) of embryonic development.

Ambystoma↗
PubMed1998

Ectopic expression of tropomyosin promotes myofibrillogenesis in mutant axolotl hearts.

Expression of tropomyosin protein, an essential component of the thin filament, has been found to be drastically reduced in cardiac mutant hearts of the Mexican axolotl (Ambystoma mexicanum) with no formation of sarcomeric myofibrils. Therefore, this naturally occurring cardiac mutation is an appropriate model to examine the effects of delivering tropomyosin protein or tropomyosin cDNA into the deficient tissue. In this study, we describe the replacement of tropomyosin by using a cationic liposome transfection technique applied to whole hearts in vitro. When mouse alpha-tropomyosin cDNA under the control of a cardiac-specific alpha-myosin heavy chain promoter was transfected into the mutant hearts, tropomyosin expression was enhanced resulting in the formation of well-organized sarcomeric myofibrils. Transfection of a beta-tropomyosin construct under control of the same promoter did not result in enhanced organization of the myofibrils. Transfection of a beta-galactosidase reporter gene did not result in the formation of organized myofibrils or increased tropomyosin expression. These results demonstrate the importance of alpha-tropomyosin to the phenotype of this mutation and to normal myofibril formation. Moreover, we have shown that a crucial contractile protein can be ectopically expressed in cardiac muscle that is deficient in this protein, with the resulting formation of organized sarcomeres.

Ambystoma↗
PubMed1995

Refined physical map of the spinal muscular atrophy gene (SMA) region at 5q13 based on YAC and cosmid contiguous arrays.

The gene for the autosomal recessive neurodegenerative disorder spinal muscular atrophy has been mapped to a region of 5q13 flanked proximally by CMS-1 and distally by D5S557. We present a 2-Mb yeast artificial chromosome (YAC) contig constructed from three libraries encompassing the D5S435/D5S629/CMS-1-SMA-D5S557/D5S112 interval. The D5S629/CMS-1-SMA-D5S557 interval is unusual insofar as chromosome 5-specific repetitive sequences are present and many of the simple tandem repeats (STR) are located at multiple loci that are unstable in our YAC clones. A long-range restriction map that demonstrates the SMA-containing interval to be 550 kb is presented. Moreover, a 210-kb cosmid array from both a YAC-specific and a chromosome 5-specific cosmid library encompassing the multilocus STRs CATT-1, CMS-1, D5F149, D5F150, and D5F153 has been assembled. We have recently reported strong linkage disequilibrium with Type I SMA for two of these STRs, indicating that the gene is located in close proximity to or within our cosmid clone array.

Base Sequence↗
PubMed1995

A recombination event occurring within two complex 5q13.1 microsatellite repeat polymorphisms suggests a telomeric mapping of spinal muscular atrophy.

The gene for the childhood spinal muscular atrophies (SMAs) has been mapped to 5q13.1. The interval containing the SMA gene has been defined by linkage analysis as 5qcen-D5S629-SMA-D5S557-5qter. We have identified a recombination event within this interval on a type-I SMA chromosome. The recombination maps to a region of multilocus microsatellite repeat (MSR) markers, and occurs between different subloci of two such markers, CMS-1 and 7613. While the possibility of a novel mutation caused by the recombination cannot be discounted, we believe when viewed in the context of a similar recombination in a Dutch SMA family, a centromeric boundary at the recombination site for the critical SMA interval is likely. This new proximal boundary would reduce the minimal region harboring the SMA locus from approximately 1.1 Mb to approximately 600 kb.

Alleles↗
PubMed1994

Two 5q13 simple tandem repeat loci are in linkage disequilibrium with type 1 spinal muscular atrophy.

The gene for the common recessive neuromuscular disorder spinal muscular atrophy (SMA) has been previously mapped to chromosome 5q. We report here linkage disequilibrium analyses of two polymorphic simple tandem repeat (STR) sequences which map into the critical region of 5q13 containing the SMA gene. The polymorphisms presented are constituents of CATT-1, a complex STR which is present in as many as four or more copies per chromosome 5. The PCR can amplify as many as eight CATT-1 products of different sizes from genomic DNA samples due to differing numbers of CA dinucleotides at each STR location (sublocus). Oligonucleotide primers for two of these subloci have been developed for specific PCR assays; a variety of allele sizes can be generated with each assay and, in some cases, no amplification products are detected due to null alleles. The genotyping of 149 SMA Type 1 chromosomes and 142 normal chromosomes from Canadian and American kindreds reveals the presence of significant linkage disequilibrium between the null allele of the sublocus referred to as CATT-40G1 and mutation(s) causing SMA Type 1 (Werdnig-Hoffmann disease). Allele 2 of the second sublocus, CATT-192F7, is also in linkage disequilibrium with SMA Type 1 although the degree of this association is less than that found for CATT-40G1. The proximal and distal STRs from the critical region, D5S435 and D5S351, showed no linkage disequilibrium with SMA. The data presented here will serve as a framework for future linkage disequilibrium analyses, expediting the final stage of the search for the SMA gene.

Alleles↗
PubMed1988

Recombination within a subclass of restriction fragment length polymorphisms may help link classical and molecular genetics.

Restriction fragment length polymorphisms (RFLPs) are being used to construct complete linkage maps for many eukaryotic genomes. These RFLP maps can be used to predict the inheritance of important phenotypic loci and will assist in the molecular cloning of linked gene(s) which affect phenotypes of scientific, medical and agronomic importance. However, genetic linkage implies very little about the actual physical distances between loci. An assay is described which uses genetic recombinants to measure physical distance from a DNA probe to linked phenotypic loci. We have defined the subset of all RFLPs which have polymorphic restriction sites at both ends as class II RFLPs. The frequency of class II RFLPs is computed as a function of sequence divergence and total RFLP frequency for highly divergent genomes. Useful frequencies exist between organisms which differ by more than 7% in DNA sequence. Recombination within class II RFLPs will produce fragments of novel sizes which can be assayed by pulsed field electrophoresis to estimate physical distance in kilobase pairs between linked RFLP and phenotypic loci. This proposed assay should have particular applications to crop plants where highly divergent and polymorphic species are often genetically compatible and thus, where class II RFLPs will be most frequent.

Chromosome Mapping↗
PubMed1983

The influence of metronidazole prophylaxis and the method of closure on wound infection in non-perforating appendicitis in childhood.

Prospective investigation of consecutive children suffering from non-perforating appendicitis indicated that metronidazole prophylaxis significantly reduces the risk of postoperative wound sepsis regardless of the method of closure. However, in view of the advantages of subcuticular polyglycolic acid this must be regarded as the method of closure of choice in non-perforating appendicitis in children.

Appendectomy↗