Sandya Narayanswami

Cytological and molecular characterization of centromeres in Mus domesticus and Mus spretus

We have applied EM in situ hybridization (EMISH) and pulsed field gel electrophoresis (PFGE) to samples from diploid primary cell cultures and an established cell line to examine in detail the relative organization of the major and minor satellite DNAs and telomere sequences in the genomes of Mus domesticus and Mus spretus. EMISH localizes the Mus domesticus minor satellite to a single site at the centromere-proximal end of each chromosome. Double label hybridizations with both minor satellite and telomere probes show that they are in close proximity and possibly are linked. In fact, PFGE of M. domesticus DNA digested with Sal I and Sfi I reveals the presence of fragments which hybridize to both probes and is consistent with the physical linkage of these two sequences. The M. domesticus minor satellite is the more abundant satellite in Mus spretus. Its distribution in M. spretus is characterized by diffuse labeling with no obvious concentration near chromosome ends. In addition to this repeat the M. spretus genome contains a small amount of DNA that hybridizes to a M. domesticus major satellite probe. Unlike the M. domesticus minor satellite, it is not telomere proximal but is confined to a domain at the border of the centromere and the long arm. Thus, although both species possess all three sequences, except for the telomeres, their distribution relative to one another is not conserved. Based on the results presented, we propose preliminary molecular maps of the centromere regions of Mus domesticus and Mus spretus.

Chromosomal location of a major tRNA gene cluster of Xenopus laevis

In Xenopus laevis, genes encoding tRNAPhe, tRNATyr, tRNA1Met, tRNAAsn, tRNAAla, tRNALeu, and tRNALys are clustered within a 3.18-kb (kilobase) fragment of DNA. This fragment is tandemly repeated some 150 times in the haploid genome and its components are found outside the repeat only to a limited extent. The fragment hybridizes in situ to a single site very near the telomere on the long arm of one of the acrocentric chromosomes of the group comprising chromosomes 13–18. All the chromosomes of this group also hybridize with DNA coding for oocyte-specific 5S RNA. The tRNA gene cluster is slightly proximal to the cluster of 5S RNA genes.

An expanded collection of mouse Y Chromosome RDA clones

If we are to understand the structure and function of the mammalian genome and its transmission to subsequent generations, we cannot neglect the Y Chromosome (Chr). However, certain properties of the Y have made it refractory to analysis. The bulk of the Y Chr does not recombine, and thus genetic maps based on standard Mendelian recombination are not feasible. For this reason, investigators have relied on physical approaches to map the human Y (Foote et al. 1992; Vollrath et al. 1992). We are using physical approaches to further our understanding of the mouse Y Chr by developing chromosome-specific DNA-based probes.

DNA sequence mapping using electron microscopy

DNA sequences can be mapped on chromosomes at high resolution in the electron microscope after hybridization with a nonisotopically labeled probe followed by detection with a two-step antibody reaction employing a colloidal gold tag. Hybridization probes can be modified with biotin-dUTP, digoxigenin-dUTP, dinitrophenyl-dUTP, or N-acetoxy-2-acetylaminofluorene (AAF). The availability of different sizes of colloidal gold particles permits the simultaneous detection of several sequences. In addition, low signals can be amplified either with an antibody sandwich scheme or by silver intensification. This technology is applicable both to TEM and SEM preparations of chromosomes, and we have used it to map a number of highly and moderately repeated sequences on whole mount metaphase chromosomes.

Chromosomal locations of major tRNA gene clusters of Xenopus laevis.

In Xenopus laevis eight tRNA genes are located in a 3.18 kb tandemly repeated unit. There are 150 copies of the unit at a single locus near the long arm telomere of one of the acrocentric chromosomes in the 14–17 group. Two additional classes of tRNA gene-containing repeats have been isolated (defined by clones p3.1 and p3.2) that have structures related to that of the 3.18 kb unit. Using in situ hybridization at the electron microscopic level, the p3.2 repeats are found clustered at a single locus in the subtelomeric region on one of the submetacentric chromosomes, whereas the p3.1 repeats are clustered at a locus indistinguishable from that containing the 3.18 kb repeats. This suggests that these tDNA tandem repeats can diverge in sequence from each other without being at distantly separated loci.

Chapter 5 Nucleic Acid Sequence Localization by Electron Microscopic in Situ Hybridization

Publisher Summary This chapter discusses the electron microscopic in situ hybridization (EMISH) technique for nucleic acid sequence localization. In situ hybridization is a pivotal genome mapping technique that provides the cytological location of a cloned sequence. The development of equivalent mapping techniques at the electron microscope (EM) level present the opportunity to determine the relative map positions of sequences. The EM localization is well suited for mapping sequences on small chromosomal structures and for subnuclear localization in small nuclei, such as yeast. It uses biotin-substituted probes and immunogold tagging of hybrid sites. EMISH is applied successfully to the localization of DNA and RNA sequences in both whole-mount metaphase chromosomes and nuclei in organisms from yeast to man. The methodology is analogous to that used at the light microscope (LM) level with a few modifications. The main source of background in EMISH appears to be the nonspecific binding of antibodies to grid films. In addition to modifying EMISH, the use of primer extension in the presence of biodUTP after hybridization of an oligonucleotide to chromosome preparations is being investigated.