Norman Arnheim - US grants

We are studying the molecular structure and genetic behavior of mouse and human ribosomal genes using recombinant DNA technology and restriction enzyme mapping. Our goal is to determine what factors influence the genetic behavior of this multichromosomally distributed repeated gene family.

Novel Approaches to Studying Mammalian Hot Spots of Recombination 1. We will continue our analysis of the molecular nature of the recombination hot spot found in the human beta globin gene complex and initiate studies on the hot spot found in the mouse immune response region using our yeast meiosis system. The goal of these investigations is to define the molecular nature of recombination hot spots at the nucleic acid level. 2. Applying a novel technology we recently developed for enzymatically amplifying DNA sequences we aim to determine the restriction fragment length polymorphism genotype of single sperm cells (haplotype determination) at the DNA level. The analysis of thousands of individual sperm using this system will allow us to study the actual frequency of recombination between very closely linked RFLPs without family studies and can be used to directly study human recombination hot spots as well as to derive accurate genetic maps over very short distances.

The complex interactions among macromolecular species in living cells provide a number of opportunities for evolutionary change to take place in each of the components involved in the interaction. An important feature of such molecular coevolution is that the interacting components in one evolutionary lineage can evolve along a pathway which differs from the same components in another species. This could lead to adaptively significant differences among species in the functional properties of the particular molecular system. An investigation of the functional divergence that has arisen among species in the system responsible for making the 18s and 28s ribosomal RNAs in mammals is proposed. It has been found that the nuclear transcriptional machinery from one species is often not capable of interacting with the DNA regulatory sequences involved in controlling ribosomal RNA gene expression from other species. The rate of evolutionary change in the transcription factors and the controlling elements of these genes will be studied. The analysis of this model system may be especially significant since it involves the analysis of evolutionary changes in elements affecting the control of gene expression. Such changes have been proposed by many to be at the basis of the characteristic differences in physiology, morphology and biochemistry that have accumulated among species during evolution.

The Polymerase Chain Reaction will be used to analyze DNA sequences in single human sperm. The ability to study large numbers of meiotic products from single individuals will allow the construction of human genetic maps at a resolution which far exceeds that possible by using conventional pedigree analysis. Sperm will be isolated using flow cytometry. The efficiency of sperm lysis and PCR amplification will be maximized under conditions which minimize contamination. The immediate goal is to be able to routinely achieve a level of resolution for genetic mapping of less than 0.1 cM (0.001 recombination fraction). Accomplishment of this goal will allow three point crosses to be used to order very closely linked markers. The relationship between physical distance and recombination frequency could also be investigated. The development of this single sperm technology may have a significant impact on mapping the human genome.

As the human genome project progresses, the detailed structural information obtained will open up many opportunities for attacking significant biological problems that were impossible to study previously. We are interested in using the techniques of single molecule PCR to study the pattern of recombination along human chromosomes, and to study the rate of specific mutations which cause human disease in the male germ line. Using information on the physical distance between DNA polymorphisms, and the method of sperm typing, the patterns of recombination potential along a large chromosomal segment will be determined and the size of the chromosomal regions which have recombination hot spot activity will be accurately defined. The effect that structural rearrangements at the human MHC have on altering the patterns of genomic recombination will also be examined. Finally, methods to detect and quantify the level of specific mutations in the male germ line will be developed.

This proposal aims to investigate the origin and mechanism of accumulation of mitochondrial DNA damage in human tissues during aging. In one case a specific mitochondrial DNA deletion previously found only in patients affected with certain rare neuromuscular diseases but recently discovered in hearts and brains of normal aged individuals will be studied. The goal of this research is to determine the rate these deletions accumulate and why they accumulate to different levels in different brain regions in aged individuals. These studies may provide information on the origin of mitochondrial mutations and their contributions to aging.

This research will continue, in several new directions, the present research program with the goal of gaining a detailed understanding of the molecular mechanisms of the transposition process, and its control, specifically of insertion sequence IS1. Insights gained to date put us in a position to answer very specific questions about the essential conponents of the transposition complex, the control of gene expression in IS1, and the regulation of transposition itself. The program of research has the additional long range goal of gaining a mechanistic understanding of the full range of recombinational events induced by the insertion sequence IS1, the molecular processes by which they occur, and the mechanisms for their regulation. Several basic molecular processes that are central to IS1 transposition promise to be novel and important. Among these are the set of key molecular events in transposition and control of gene expression that take place at the ends of IS1 (all within about 30 base-pairs), and the possibility that translational frameshifting is a critical regulatory event that takes place in the center of IS1. These mechanisms, appear to be different from those of other well-studied transposable elements like TnlO, Tn3, Tn7, Mu phage, IS5 and IS50. IS1 is one of the most widespread of transposable elements, and elucidation of the basic molecular mechanisms is essential to an understanding of the IS-mediated assembly of genes into plasmids and dissemination of genetic information in the bacterial world. The proposed project aims to study the apparent frameshifting that produces a fused InsA-B protein, which may be the ISI transposase. All the IS1-encoded components of the transposition complex, essential or accessory, will be characterized. The binding properties of the repressor (InsA) and the (putative) transposase will be studied via mutational analysis, of both the proteins and the DNA sites, and by physical methods. A new genetic system for the study of IS1 will be developed, which permits selection and screening for mutants in the transpostion pathway. Finally, new results with the fusion protein will be used to develop an in vitro system for IS1 transpostion, which will be used to dissect the process biochemically. Detailed molecular models of IS1 transpostion will be contructed from these results and tested further.

This proposal seeks to study fundamental questions concerning mutation and recombination events that lead to human disease. One aim is to determine whether expansions of the CAT/CTG tracts found in Huntington disease patients occur in germline mitotic cells or following the initiation of meiosis. Studies are also proposed to examine whether the proximity or orientation of a CAG/CTG tract relative to an origin of DNA replication influences expansion size or frequency and whether this accounts for the marked inter-locus variation in expansion mutation susceptibility. Two other aims also focus on mutation. One aim will examine human sperm to determine whether the mutation that causes achondroplasia, the most common cause of dwarfism, increases with the age of the father as predicted by population studies. Another will examine the role played by members of the MutL DNA repair protein family on mononucleotide repeat slippage mutations. In humans this kind of mutation has been shown to inactivate important genes in many tumors from patients with the a familial colon cancer (HNPCC). The last aim seeks to directly measure the effects of sequence length and sequence similarity on the frequency of unequal recombination between repeated sequences in the human genome. Such events have been shown to lead to a variety of human disease syndromes.