Developmental genomics and its relation to aging.
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Aging↗
Biomedical subjects
M S Ko
Publications and source records attributed to M S Ko.
Tissue-specific expression and mapping of the Cox7ah gene in mouse.
We have isolated and examined the gene for the heart isoform of cytochrome c oxidase subunit VIIa (COX VIIa-H) in mouse, an isoform gene previously thought to be lacking in rodents. Interspecies amino acid comparisons indicate that mouse COX VIIa-H protein displays 82.5 and 70.9% identity with the bovine and human heart isoforms of COX VIIa, but only 53.7% identity with the paralogous mouse liver isoform (COX VIIa-L). Expression in adult mouse tissues is limited to heart and skeletal muscle, as found in other species. In the early mouse embryo, Cox7al was the exclusive isoform expressed and Cox7ah mRNA was not detectable until day 17 postcoitum. That the mouse Cox7ah gene characterized in this study is orthologous to the human COX7AH gene was also suggested by its mapping to mouse chromosome 7, to a conserved region syntenic with the human chromosome location of COX7AH, 19q13.1. As a result, all three COX heart isoform genes in mouse group to chromosome 7. Interestingly, mapping of the mouse Cox7al to chromosome 9 suggests a new syntenic region between the mouse and the human genomes.
Amino Acid Sequence↗
Genome-wide mapping of unselected transcripts from extraembryonic tissue of 7.5-day mouse embryos reveals enrichment in the t-complex and under-representation on the X chromosome.
Mammalian embryos can only survive if they attach to the uterus (implantation) and establish proper maternal-fetal interactions. To understand this complex implantation pathway, we have initiated genomic analysis with a systematic study of the cohort of genes expressed in extraembryonic cells that are derived from the conceptus and play a major role in this process. A total of 2103 cDNAs from the extraembryonic portion of 7.5-day post-conception mouse embryos yielded 3186 expressed sequence tags, approximately 40% of which were novel to the sequence databases. Furthermore, when 155 of the cDNA clones with no homology to previously detected genes were genetically mapped, apparent clustering of these expressed genes was detected in subregions of chromosomes 2, 7, 9 and 17, with 6.5% of the observed genes localized in the t-complex region of chromosome 17, which represents only approximately 1.5% of the mouse genome. In contrast, X-linked genes were under-represented. Semi-quantitative RT-PCR analyses of the mapped genes demonstrated that one third of the genes were expressed solely in extraembryonic tissue and an additional one third of the genes were expressed predominantly in the extraembryonic tissues. The over-representation of extraembryonic-expressed genes in dosage-sensitive autosomal imprinted regions and under-representation on the dosage-compensated X chromosome may reflect a need for tight quantitative control of expression during development.
Animals↗
A systematic molecular genetic approach to study mammalian germline development.
It is difficult to study gene expression in mammalian embryonic germ cells as PGCs constitute only a minor proportion of the mouse embryo. We have overcome this problem by using a novel combination of established molecular and transgenic approaches. A line of mice has been generated in which the cells of the germ lineage express the beta-galactosidase reporter gene during embryogenesis. Using this line, germ cells have been purified to near homogeneity from embryos at discrete stages during germline development by use of a stain for beta-gal activity and a fluorescence activated cell sorter. Subsequently, cDNA libraries have been constructed from each germ cell population using a modified lone-linker PCR strategy. These combined cDNA libraries represent genes expressed in PGCs during mammalian germline development. To facilitate a molecular genetic approach to studying mammalian germline development, these cDNA libraries will be pooled to form an arrayed, addressed reference embryonic germ cell cDNA library. In parallel with large-scale cDNA sequencing efforts; genes that are differentially expressed in germ cells will be identified by screening the reference library with probes generated by subtractive hybridization. Complementary DNAs identified using this approach will be analyzed by sequencing, database comparison, genomic mapping and in situ hybridization to ascertain the potential functional importance of each gene to germline development. In addition to providing a wealth of novel information regarding patterns of gene expression during mammalian germline development, these results will form the basis for future experiments to determine the function of these genes in this process.
Animals↗
The Tabby phenotype is caused by mutation in a mouse homologue of the EDA gene that reveals novel mouse and human exons and encodes a protein (ectodysplasin-A) with collagenous domains.
Mouse Tabby (Ta) and X chromosome-linked human EDA share the features of hypoplastic hair, teeth, and eccrine sweat glands. We have cloned the Ta gene and find it to be homologous to the EDA gene. The gene is altered in two Ta alleles with a point mutation or a deletion. The gene is expressed in developing teeth and epidermis; no expression is seen in corresponding tissues from Ta mice. Ta and EDA genes both encode alternatively spliced forms; novel exons now extend the 3' end of the EDA gene. All transcripts recovered have the same 5' exon. The longest Ta cDNA encodes a 391-residue transmembrane protein, ectodysplasin-A, containing 19 Gly-Xaa-Yaa repeats. The isoforms of ectodysplasin-A may correlate with differential roles during embryonic development.
Alternative Splicing↗
Differential expression pattern of XqPAR-linked genes SYBL1 and IL9R correlates with the structure and evolution of the region.
The recently discovered second pseudoautosomal region (XqPAR) contains at least two genes, IL9R and SYBL1. Recent findings show that, like XpPAR genes, IL9R escapes X inactivation and its Y allele is also expressed, but SYBL1 seems to act like an X-linked gene, expressed from the active X chromosome but not from the inactive X or Y. Here we show that differences are also seen in the evolution of the sex chromosome locations of IL9R and SYBL1. IL9R is known to be autosomal in mice, and is X-linked only in primates. SYBL1, however, has been found to be on the X chromosome in all mammals tested, from marsupials to humans. Both genes were duplicated on the Y homologue of the terminal portion of the X chromosome during the evolution of Homo sapiens from other higher primates. The inactivation pattern of SYBL1 may be correlated with its longer history of X linkage, and at a more centromeric chromosomal position during evolution; the more recent X linkage and more telomeric position of the IL9R gene may explain its autosomal, 'uninactivated' transcriptional status.
Animals↗
Basal cell carcinoma occurring in multiple familial trichoepithelioma: detection of loss of heterozygosity in chromosome 9q.
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Aged↗
Cloning and expression analyses of mouse dystroglycan gene: specific expression in maternal decidua at the peri-implantation stage.
While constructing a catalog of mouse cDNAs which are expressed in the maternal-fetal interface during the peri-implantation period, we encountered a 1.6 kb cDNA clone showing a strong sequence similarity to the 3' untranslated region of the human dystroglycan gene. We cloned an additional 1.7 kb cDNA by reverse transcriptase-PCR (RT-PCR) and confirmed that this is a true mouse homolog of human dystroglycan cDNA by sequence analyses, Southern blotting, and genetic mapping of this gene on the distal region of mouse chromosome 9. Although it is well established that dystroglycan, a transmembrane protein, plays an important role in muscle tissues by bridging intracellular dystrophin to the laminin in the extracellular matrix, its role in non-muscle tissues remains elusive. To further investigate the role of the dystroglycan gene at the peri-implantation stage, we analyzed the expression patterns of this gene by in situ hybridization, which revealed that this gene is specifically expressed in decidual cells, especially in the cells surrounding the implantation site at 6.5, 7.5, and 8.5 day post conception (p.c.) stages, but not expressed in non-pregnant endometrial cells of uterus nor in the decidua at 12.5 day p.c. Further analyses by RT-PCR confirmed that the amount of dystroglycan mRNA in 8.5 day p.c. decidua was indeed 100-fold higher than that of non-pregnant uterus and 12.5 day p.c. mature placenta. These results suggest that dystroglycan may work as a mediator for adhesion between decidual cells themselves or between decidual cells and trophoblast cells, and provide a structural and functional support for maintaining pregnancy at its early stage.
Amino Acid Sequence↗
The gene for multiple familial trichoepithelioma maps to chromosome 9p21.
Multiple familial trichoepithelioma (MFT) is an autosomal dominant skin disease characterized by the presence of many small tumors predominantly on the face. To map the causative gene, we performed linkage analysis with microsatellite markers in three American families. We found a significant linkage of a gene for MFT to chromosome 9p2l. The maximum combined lod score was 3.31 at D9S171 at theta = 0. The disease locus was defined to a 4-cM region between IFNA and D9S126. Because several tumor suppressor genes including p16 and p15 have been mapped to this region, the gene for MFT may also be a tumor suppressor.
Chromosome Mapping↗
Toward a whole cDNA catalog: construction of an equalized cDNA library from mouse embryos.
The construction of a cDNA library containing all genes without redundancy is one of the major technical challenges for biology. Toward this goal, we have developed an equalized (normalized) cDNA library from mRNA pools derived from mouse embryos that cover the entire period of mouse ontogenesis. Colony hybridization analyses with 11 genes showed the reduction of abundance variation from at least 6000-fold in the unequalized S-library to approximately 33-fold in the EIII-library, which was constructed after three cycles of equalization procedure. Limiting dilution PCR analyses with 26 tissue-specific genes showed the reduction of abundance variation from at least approximately 1,000,000-fold in the S-cDNA mixture to approximately 100-fold in the EIII-cDNA mixture. Based on these analyses, we estimate that at least 15,000 independent cDNA clones are included with little redundancy in the EIII-cDNA library. This will be a useful resource for mouse biology as well as the mouse genome project.
Animals↗
Genetic mapping of 40 cDNA clones on the mouse genome by PCR.
We recently proposed a new PCR-based genetic marker assay for the mouse genome that exploits sequence differences in the 3'-untranslated region (UTR) of cDNAs between different mouse strains, called "biallelic polymorphic expressed sequence tags (bESTs)." The specific use of 3'-UTR has several advantages: (1) frequent sequence polymorphism between different mouse strains, (2) most commonly uninterrupted by introns, (3) usually unique sequence even among closely related gene family members. In this paper, we identify additional genetic loci defined by bEST and determine their location on the mouse genetic map by using interspecific backcross mapping panels between C57BL/6J and Mus spretus. Of 136 markers tested, 86 produced unique PCR products from C57BL/6J and M. spretus genomic DNAs. We then sequenced these 86 PCR products from C57BL/6J and M. spretus and found that 59 markers have sequence polymorphisms. Of these, we mapped 36 by restriction fragment length polymorphism (RFLP) of the PCR products and 4 by length polymorphism (LP) of the PCR products. We discuss the possibility of a large-scale application of this method for cDNA mapping.
Animals↗
Maps from two interspecific backcross DNA panels available as a community genetic mapping resource.
We established two mouse interspecific backcross DNA panels, one containing 94 N2 animals from the cross (C57BL/6J x Mus spretus)F1 x C57BL/6J, and another from 94 N2 animals from the reciprocal backcross (C57BL/6J x SPRET/Ei)F1 x SPRET/Ei. We prepared large quantities of DNA from most tissues of each animal to create a community resource of interspecific backcross DNA for use by laboratories interested in mapping loci in the mouse. Initial characterization of the genetic maps of both panels has been completed. We used MIT SSLP markers, proviral loci, and several other sequence-defined genes to anchor our maps to other published maps. The BSB panel map (from the backcross to C57BL/6J) contains 215 loci and is anchored by 45 SSLP and 32 gene sequence loci. The BSS panel map (from the backcross to SPRET/Ei) contains 451 loci and is anchored by 49 SSLP loci, 43 proviral loci, and 60 gene sequence loci. To obtain a high density of markers, we used motif-primed PCR to "fingerprint" the panel DNAs. We constructed two maps, each representing one of the two panels. All new loci can be located with a high degree of certainty on the maps at current marker density. Segregation patterns in these data reveal several examples of transmission ratio distortion and permit analysis of the distribution of crossovers on individual chromosomes.
Animals↗
Simple and robust screening of pooled yeast artificial chromosome libraries by the restriction enzyme digestion of polymerase chain reaction products.
The polymerase chain reaction (PCR)-based method for screening pooled yeast artificial chromosome (YAC) libraries was modified by adding a step for the restriction enzyme digestion of the PCR products. This modification significantly increased the reliability of YAC screening and made it possible to identify YAC clones without a cumbersome verification step by colony hybridization and/or Southern blotting. Using this method, we assigned 39 mouse YAC clones to a mouse genetic map with 13 biallelic, polymorphic expressed sequence tags. This method provides a fast, reliable way to identify YAC clones with PCR-based sequence-tagged sites.
Animals↗
Optimized conditions for cycle sequencing of PCR products.
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DNA Primers↗
The short 3'-end region of complementary DNAs as PCR-based polymorphic markers for an expression map of the mouse genome.
Ideal markers for a complementary DNA (cDNA) map of the mouse genome should be amplifiable by the polymerase chain reaction (PCR) and they should be polymorphic for genetic mapping, as well as unique for physical mapping. In our search for such markers, we did comparative sequencing of PCR-amplified genomic DNAs derived from 15 inbred strains and found that about 250 bp of the 3'-end region (3'-ER) of cDNAs, which is the sequence immediately upstream from the poly(A) tail, was polymorphic enough to distinguish the allele of a laboratory strain from that of a wild strain (Mus spretus) in 14 of 22 cDNAs tested. Specifically, in 9 of these 14 cDNAs, each allele was identified by the restriction fragment length polymorphism. This data indicates that about 65% of the 3'-ERs of cDNAs can be automatically transformed into PCR-based genetic markers named "biallelic polymorphic expressed sequence tags (bESTs)." These markers can be easily and precisely mapped by typing of the panels of interspecific backcrosses. Because a large number of bEST markers can be efficiently obtained by a single-run automated sequencing of randomly selected cDNAs, these markers will greatly facilitate the construction of high-resolution genetic and physical maps of expressed sequences of the mouse genome.
Alleles↗
[An equalized cDNA library and its application].
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Animals↗
Induction mechanism of a single gene molecule: stochastic or deterministic?
A new field of gene expression regulation research is emerging that has previously been overlooked. This new area is concerned with distinguishing the expression of a single gene from the averaged expression of many gene copies within the cell population. This paper reviews research focused on individual genes in inducible gene expression systems. The main experimental strategy is to measure the gene expression level of a single cell containing a single reporter gene molecule. In contrast to the commonly held belief, gene induction is found to be stochastic under certain conditions. The possible mechanisms and implications are discussed.
Gene Expression Regulation↗
A stochastic model for gene induction.
Expression levels of individual copies of an inducible gene have been presumed to be identical to the averaged level of many copies and to change in a smooth and predictable way according to the concentration of an inducing molecule. However, our recent experiments using a steroid-inducible system showed that the expression levels of individual copies are very heterogeneous and do not necessarily coincide with the averaged expression level of many copies (Ko et al., 1990, EMBO J. 9, 2835-2842). To explain this result, I present a stochastic model for gene induction here and its analysis using computer simulation. Stochasticity in the model is derived from the randomness corresponding to the random timing of molecular collisions and dissociations between transcription factors and a gene copy, since at any instant each copy is thought to be either "switched on" by having a transcription complex bound to it, or "switched off" by not having a transcription complex bound. This model can produce two types of gene induction that depend on the stability of the transcription complex on the regulatory region of the gene. An unstable transcription complex causes a homogeneous level of gene induction among individual copies, while a stable transcription complex causes a heterogeneous level. Since the recent consensus formed by in vitro transcription experiments is that the transcription complex is generally very stable, the latter case (the non-deterministic one) is highly possible. Since typical eukaryotic cells have just two copies for any gene in a single cell, this possibility of heterogeneous gene induction indicates that the phenotypes of individual cells cannot be precisely determined by just environmental signals, such as inducers. This may prompt us to reconsider many problems related to gene induction, including morphogenesis.
Animals↗