Mechanisms of Eukaryotic DNA Recombination

Max E Gottesman, Henry J. Vogel, Max E Gottesman, Henry J. Vogel

• Publisher: Academic Press
• Year: 2014
• Print ISBN: 9780122934452
• eBook ISBN: 9781483274089
• Topic: Biological Sciences
• Subtopic: Biology
• Index: Biological Sciences

Mechanisms of Eukaryotic DNA Recombination is a collection of papers that discusses advances in eukaryotic genetic recombination. Papers address issues in eukaryotic genetic recombination, particularly DNA integration in mammalian genomes, genetic recombination in Drosophila or Caenorhabditis; the manipulation of the mouse genome; genome organization; and genetic recombination in protozoa. One paper discusses chromatid interactions during intrachromosomal recombination in mammalian cells, namely, intrachromatid and sister chromatid. Another paper analyzes the implication for chromosomal recombination and gene targeting; results on extrachromosomal recombination show that circles are inefficient substrates for recombination even if only one of two substrates in an intermolecular reaction is circular. One author discusses the genetics and molecular biology of recombination, citing the work of Watson and Crick, stating that crossing-over occurs between genes (not within them). He also explains that the formation and resolution of recombination intermediaries depend on enzyme or other proteins. This book will prove invaluable to cellular biologists, microbiologists, and researchers engaged in genetics and general biology.

PART I

TARGETED DNA INTEGRATION IN MAMMALIAN CELLS

Outline

Chapter 1: Chromatid Interactions during Intrachromosomal Recombination in Mammalian Cells

Chapter 2: Studies on Extrachromosomal Homologous Recombination in Mammalian Cells: Implications for Chromosomal Recombination and Gene Targeting

Chapter 3: Homologous Recombination in Embryonic Stem Cells as a Means to Generate Mice with Defined Mutations

Chapter 4: Identification and Targeted Mutation of Developmental Genes in Mouse Embryonic Stem Cells

1

Chromatid Interactions during Intrachromosomal Recombination in Mammalian Cells

RONI J. BOLLAG and R. MICHAEL LISKAY, Departments of Human Genetics and Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06510

Publisher Summary

This chapter discusses chromatid interactions during intrachromosomal recombination in mammalian cells. Intrachromosomal recombination can involve two discrete types of chromatid interactions: (1) intrachromatid, which occurs between linked sequences on a DNA molecule and (2) sister chromatid, which occurs between equally or unequally paired homologous sequences following DNA replication. In most cell systems, equal sister chromatid exchange has no genetic consequence. Orientation I refers to a configuration in which mutations are proximal to the intervening sequences, whereas the orientation II constructs harbors genes with mutations distal to the intervening sequences. In the study presented in the chapter, the directions of both tk genes were reversed on the recombination substrate to produce direct repeats in orientation II, and the construct was introduced into mouse L cells by direct nuclear microinjection. The focus of the study was a comparison of products of reciprocal recombination from orientation I and orientation II direct repeats. The primary goal is to infer the types of chromatid interactions among repeated genes. Because gene conversions are noninformative, the strategy involves a comparison of reciprocal exchanges between orientation I and orientation II direct repeats.

INTRODUCTION

Intrachromosomal recombination can involve two discrete types of chromatid interactions: intrachromatid, which occurs between linked sequences on a DNA molecule and sister chromatid, which occurs between equally or unequally paired homologous sequences following DNA replication (see Fig. 1). In most cell systems, equal sister chromatid exchange has no genetic consequence.

Fig. 1 Types of chromatid interactions. Diagrammed are sister chromatids with pairs of repeated genes (blocks). (1) Intrachromatid exchange between linked genes on one chromatid. Interactions of this type can occur at the two-strand or at the four-strand stage. (2) Unequal sister chromatid exchange between genes at nonallelic positions on sister chromatids. Such interactions require unequal pairing. (3) Equal sister chromatid exchange between identical sequences at allelic positions. Such interactions, if they occur, have no genetic consequence in recombination studies.

Gene conversion is the predominant mode of intrachromosomal recombination in mouse L cells (1). Since such nonreciprocal exchanges involve unidirectional information transfer between DNA molecules, sister chromatid and intrachromatid convertants appear identical and do not impart information concerning the interaction involved. On the other hand, reciprocal exchanges often lead to distinct products in which the type of interaction can be ascertained. For instance, intrachromatid reciprocal exchange between genes in an inverted orientation generates inversion products, whereas sister chromatid exchanges lead to inviable aberrant products that are not recovered (2). Simple intrachromatid reciprocal exchange between direct repeats should generate two products: a circular DNA molecule with one gene and a single hybrid gene located in the chromosome and lacking the sequence between the two interacting sequences. Sister chromatid reciprocal exchange between a pair of direct repeats gives rise to one chromatid with a triplication and one chromatid with only a single gene and deletion of the intervening sequence. During cell propagation without selection, a circular DNA molecule should be lost. Chromatids with only a single gene are designated deletion products in this study. Deletion products of intrachromatid exchange are indistinguishable from deletion products of sister chromatid exchange. In the tk-selective system used in the present study, the reciprocal product that would harbor a wild-type gene would depend on the orientations of mutations within the genes (see Figs. 2 and 3). The outcomes of analyses of these products form the basis of this study.

Fig. 2 Simple reciprocal recombinations between direct repeats in orientation I. Mutations are proximal to intervening sequence. Open blocks designate Xho I linker insertion mutations tk26 or tk8. Top panel depicts intrachromatid exchange. H refers to HindIII restriction sites flanking the tk26 allele. B refers to BamHI restriction sites flanking tk8 allele. Simple reciprocal exchange in the region between the mutations leads to a wild-type sequence in the chromosome with looping out of the double mutant gene and neo. Reciprocal products have hybrid flanking markers, H and B. Bottom panel depicts sister chromatid exchange. Centromere location is hypothetical; the relative orientation of the centromere is inconsequential. Simple reciprocal exchange between tk8 and tk26 alleles on either strand generates a single wild-type gene on one chromatid, and a double-mutant gene flanked by two mutant genes on the sister chromatid, which is lost. Deletion products of either intrachromatid or sister chromatid exchange are indistinguishable and ...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Preface
• PART I: TARGETED DNA INTEGRATION IN MAMMALIAN CELLS
• PART II: DNA INSERTION PHENOMENA
• PART III: GENETIC RECOMBINATION IN DROSOPHILA AND CAENORHABDITIS
• PART IV: GENETIC RECOMBINATION IN YEASTS AND USTILAGO
• PART V: GENETIC RECOMBINATION IN TRYPANOSOMES AND PLASMODIUM
• Index