Genetic Fine Structure Analysis Research Articles

Finding meaning in the cover design

This book is intended to describe the basic principles that underlie genetic analysis in a manner that could and should be understood by those undertaking an advanced course. It is much more compact than a previous book on this subject at just fewer than 240pp (the previous tome by Suzuki et al, being a more meaty 600+pp). However, brevity notwithstanding, I found this to be a well thought out and agreeably presented book. The subject matter follows in a logical progression from types of mutation, mutants and how to find them, then onto the array of organisms or 'Gallery of Model Organisms' one can use. In this section each species is concisely introduced but links are provided both to appropriate URLs and further reading, allowing an informed choice to be made. The complementation test and suppression are covered in some detail followed by chapters on epistasis and gene function then genetic fine-structure analysis. Finally the book is concluded with two chapters on recombination and chromosome segregation.

Analysis of recombination sites within the maize waxy locus.

Genetic fine structure analysis of the maize wx locus has determined that the ratio of genetic to physical distance within wx was one to two orders of magnitude higher than the average for the maize genome. Similar results have been found at other maize loci. In this study, we examined several mechanisms that could account for this pattern. First, crossovers in two other maize genes resolve preferentially at specific sites. By mapping exchanges between wx-B1 and wx-I relative to a polymorphic SstI site, we found no evidence for such a hotspot at wx. Second, deletion of promoter sequences from wx alleles had little effect on recombination frequencies, in contrast to results in yeast where promoter sequences are important for initiating recombination in some genes. Third, high levels of insertion polymorphism may suppress intergenic recombination. However, the presence of a 2-kb Ds element 470 bp upstream of the wx transcription start site did not further suppress recombination between Ds insertions in nearby wx sequences. Thus, none of these mechanisms is sufficient to explain the difference between intergenic and intragenic recombination rates at wx.

X-ray-induced specific-locus mutation in the ad-3 region of two-component heterokaryons of Neurospora crassa: VI. Induction kinetics of gene/point mutations, multilocus deletions and multiple-locus mutations

Genetic fine-structure analysis of X-ray-induced specific-locus mutants in the ad-3 region of two-component heterokaryons of Neurospora crassa has shown that gene/point mutations, multilocus deletions and multiple-locus mutations are induced. When the dose-response curves for these classes of ad-3 mutants were plotted, it was demonstrated that X-ray-induced gene/point mutations ( ad-3 R) increased linearly with X-ray dose and X-ray-induced multilocus deletions increased as the square of the X-ray dose. However, all classes of multiple-locus mutations, which would be expected to result from 3 to 8 hits on the basis of target theory (Lea, 1955), were found to increase as the square of the dose. Target theory assumes that the DNA of individual chromosomes is distributed randomly throughout the interphase nucleus. A model of eukaryotic interphase chromosome structure in which the DNA of individual chromosomes presents a nonrandom target to X-rays [Pinkel et al., Proc. Natl. Acad. Sci. (U.S.A.) 83 (1986), 2934–2938] provides a possible explanation for the high frequency and dose-squared induction kinetics of the multiple-locus mutants induced by X-rays in the ad-3 region.

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa V. Irreparable mutants of genotype ad-3 Aad-3 B, ad-3 Aad-3 Bnic-2, and ad-3 Bnic-2 result from multilocus deletion and an unexpectedly high frequency of multiple-locus mutations

More extensive complementation tests than those performed initially (Webber and de Serres, 1965) on a series of 832 X-ray-induced specific-locus mutations in the adenine-3 ( ad-3) region of a two-component heterokaryon (H-12) of Neurospora crassa (de Serres, 1989a) showed that unexpectedly high frequencies of specific-locus mutations in the ad-3 region have additional, but separate, sites of recessive lethal ( RL CL ) damage in the immediately adjacent genetic regions. The frequencies of these X-ray-induced multiple-locus mutants in the ad-3 region are orders of magnitude higher than that expected on the basis of target theory and classical models of chromosome structure during interphase (de Serres, 1989a). Genetic fine structure analyses, by means of homology tests with tester strains carrying genetic markers in the ad-3 and immediately adjacent regions, have been performed to map the presumed multiple-locus mutations. In a previous paper (de Serres, 1989c), X-ray-induced irreparable ad-3 mutants of the following genotypes and numbers ( ad-3 A [29] or ad-3 B [63]) were analyzed, and the high frequency of multiple-locus mutations was confirmed. In the present paper, X-ray-induced irreparable ad-3 mutants of the following genotypes and numbers ( ad-3 A ad-3 B [90], ad-3 A ad-3 B nic-2 [10]) and ad-3 B nic-2 [4]) have also been subjected to the same genetic fine structure analysis. These experiments, in the previous (de Serres, 1989c) and present papers, were designed to determine the extent of the functional inactivation in the ad-3 and immediately adjacent genetic regions in individaul mutants classified as presumptive multilocus deletions or multiple-locus mutations. The data in the present paper have shown that in Neurospora crassa most X-ray-induced irreparable mutants of genotype ad-3 A ad-3 B, ad-3 A ad-3 B nic-2, and ad-3 B nic-2 map as a series of overlapping multilocus deletions. Among the mutants of genotype ad-3 A ad-3 B, there were 34 different subgroups of complementation patterns; among those of genotype ad-3 A ad-3 B nic-2, there were 5 different subgroups; and among those of genotype ad-3 B nic-2, there were 4 different subgroups. In addition, genetic fine structure analysis has shown that some of the mutants classified, initially, as multilocus deletions ( ad-3 IR), are actually multiple-locus mutations: multilocus deletions with closely linked, and separate, sites of recessive lethal damage with a wide variety of genotypes. Combining data from the present experiments with previously published data (de Serres, 1989a,c), the frequency of multiple-locus mutations among X-ray-induced gene/point mutations and multilocus deletions in the ad-3 region is 6.2% ( 51 828 ).

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa: IV. Irreparable mutants of genotype ad-3A and ad-3B result from multilocus deletion and an unexpectedly high frequency of multiple-locus mutations

The induction of specific-locus mutations in the ad-3 region of Neurospora crassa after X-irradiation was studied in a two-component heterokaryon to determine: (1) the ratio of reparable ad-3 mutants (presumed gene/point mutations, designed ad-3 R) to irreparable ad-3 mutants (presumed multilocus deletions, designated ad-3 IR), and (2) the induction kinetics of each class (Webber and de Serres, 1965). More extensive genetic tests made subsequently (de Serres, 1989a) on the 832 X-ray-induced specific-locus mutations recovered in those experiments showed that unexpected high frequencies of reparable and irreparable ad-3 mutants are actually multiple-locus mutants that have additional, but separate, sites of recessive lethal ( RL CL) damage in the immediately adjacent genetic regions (designated ad-3 R + RL CL or ad-3 IR + RL CL). The frequencies of these X-ray-induced multiple-locus mutants in the ad-3 region are orders of magnitude higher than expected on the basis of target theory (where the frequency of the double mutant is expected to be the product of the frequencies of each single mutant) and classical models of chromosone structure during interphase (de Serres, 1989a). In the present paper, a random sample of 832 X-ray-induced ad-3 mutants of genotype ad-3A or ad-3B that are irreparable have been subjected to more extensive genetic fine-structure analysis. These experiments were designed to determine the extent of the functional inactivation in individual mutants in the ad-3 and immediately adjacent genetic regions in mutants classified as presumptive multilocus deletions or multiple-locus mutations. These experiments have shown that in Neurospora crassa most X-ray-induced irreparable mutants of genotype ad-3A or ad-3B map as a series of overlapping multilocus deletions. Among the 29 irreparable mutants of genotype ad-3A, there are 16 different subgroups of complementation patterns; and among the 63 irreparable mutants of genotype ad-3B, there are also 16 different subgroups. In addition, mutants classified as presumptive multiple-locus mutants result from a variety of separate, but closely linked, sites of genetic damage. Combining data from the present experiments with previously published data (de Serres, 1989a), the frequency of multiple-locus mutations of genotype ad-3 R + RL CL among X-ray-induced gene/point mutations ( ad3A R or ad-3B R) in the ad-3 region is 3.4% ( 22 644 ), and the frequency of multiple-locus mutations of genotype ad-3 IR + RL CL (or ad-3 IR + RL CL + RL CL) among X-ray-induced multilocus deletions ([ ad-3A ad-3B] IR, [ ad-3A ad-3B nic-2] IR) in the ad-3 region is 6.7% ( 13 195 ).

X-ray-induced specific locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa: III. Genetic fine structure analysis of the ad-3 and immediately adjacent genetic regions by means of complementation tests

Genetic fine structure analysis of the ad-3 and immediately adjacent regions was made by means of complementation tests on all possible pairwise combinations of 50 X-ray-induced irreparable adenine-3 mutants (designated ad-3 IR). All mutants were induced in either heterokaryon 11 or heterokaryon 12 of Neurospora crassa, 2-component heterokaryons heterozygous for mutants at the 3 closely linked loci ad-3A and ad-3B and nic-2 (nicotinamide-requiring) located about 5.0 map units distal to ad-3B. The complementation tests involved mutants of the following genotypes: 15 ad-3A, 27 ad-3B, 7 ad-3A ad-3B nic-2 and 1 ad-3B nic-2. To facilitate mapping, 5 additional strains (each consisting of a gene/point mutation at the ad-3A or ad-3B locus and a separate site of closely linked recessive lethal damage in the immediately adjacent regions [designated ad-3 R + RL CL]) were also included. The data from these complementation tests showed that the majority ( 46 50 ) of X-ray-induced irreparable ad-3 mutants mapped as a series of overlapping multilocus deletions that extend both proximally and distally into the immediately adjacent genetic regions, as well as into the ‘X’ region (a region of unknown, but essential function) between ad-3A and ad-3B. The remaining mutants ( 4 50 ) were found to result from a series of closely linked, but separate, mutations (designated multilocus mutations) of the type ad-3 IR + RL CL, different from those found in previous studies (de Serres, 1968; de Serres and Brockman, 1968). The data from the present complementation tests have expanded the process of genetic fine structure mapping of the ad-3 and immediately adjacent regions (de Serres, 1968) and defined the presence of the following 11 genetic loci: (a) 4 loci (with either known [i.e. col-lt] or unknown [i.e. unknA]) function proximal to ad-3A: unknA, unknB, col-lt, and col-2t, (b) 4 loci in the ‘X’ region: unknC, unknD, unknE, and unknF, (c) 2 loci distal to ad-3B: unknG, col-3t, and (d) 1 locus distal to nic2: unknH.

Highly Mutable Sites for ICR-170-Induced Frameshift Mutations Are Associated with Potential DNA Hairpin Structures: Studies with SUP4 and Other Saccharomyces cerevisiae Genes

The majority of the mutations induced by ICR-170 in both the CYC1 gene (J. F. Ernst et al. Genetics 111:233-241, 1985) and the HIS4 gene (L. Mathison and M. R. Culbertson, Mol. Cell. Biol. 5:2247-2256, 1985) of the yeast Saccharomyces cerevisiae were recently shown to be single G . C base-pair insertions at monotonous runs of two or more G . C base pairs. However, not all sites were equally mutable; in both the CYC1 and HIS4 genes there is a single highly mutable site where a G . C base pair is preferentially inserted at a [sequence in text]. Here we report the ICR-170 mutagen specificity at the SUP4-o tyrosine tRNA gene of yeast. Genetic fine structure analysis and representative DNA sequence determination of ICR-170-induced mutations revealed that there is also a single highly mutable site in SUP4-o and that the mutation is a G . C base-pair insertion at a monotonous run of G . C base pairs. Analysis of DNA sequences encompassing the regions of highly mutable sites for all three genes indicated that the mutable sites are at the bases of potential hairpin structures; this type of structure could not be found at any of the other, less mutable G . C runs in SUP4, CYC1, and HIS4. Based on these results and recent information regarding novel DNA structural conformations, we present a mechanism for ICR-170-induced mutagenesis. (i) ICR-170 preferentially binds to DNA in the beta conformation; factors that increase the temporal stability of this structure, such as adjacent stem-and-loop formation, increase the frequency of ICR-170 binding; (ii) the observed mutagen specificity reflects formation of a preferred ICR-170 intercalative geometry at [sequence in text] sites; (iii) during replication or repair, ICR-170 remains associated with the single-stranded template; (iv) stuttering or strand slippage by the polymerization complex as it encounters the mutagen results in nucleotide duplication; (v) subsequent replication or mismatch repair fixes the insertion into the genome. This mechanism accounts for both the IRC-170 mutagenic specificity and the molecular basis of the highly mutable sites in S. cerevisiae.

Highly mutable sites for ICR-170-induced frameshift mutations are associated with potential DNA hairpin structures: studies with SUP4 and other Saccharomyces cerevisiae genes.

The majority of the mutations induced by ICR-170 in both the CYC1 gene (J. F. Ernst et al. Genetics 111:233-241, 1985) and the HIS4 gene (L. Mathison and M. R. Culbertson, Mol. Cell. Biol. 5:2247-2256, 1985) of the yeast Saccharomyces cerevisiae were recently shown to be single G . C base-pair insertions at monotonous runs of two or more G . C base pairs. However, not all sites were equally mutable; in both the CYC1 and HIS4 genes there is a single highly mutable site where a G . C base pair is preferentially inserted at a [sequence in text]. Here we report the ICR-170 mutagen specificity at the SUP4-o tyrosine tRNA gene of yeast. Genetic fine structure analysis and representative DNA sequence determination of ICR-170-induced mutations revealed that there is also a single highly mutable site in SUP4-o and that the mutation is a G . C base-pair insertion at a monotonous run of G . C base pairs. Analysis of DNA sequences encompassing the regions of highly mutable sites for all three genes indicated that the mutable sites are at the bases of potential hairpin structures; this type of structure could not be found at any of the other, less mutable G . C runs in SUP4, CYC1, and HIS4. Based on these results and recent information regarding novel DNA structural conformations, we present a mechanism for ICR-170-induced mutagenesis. (i) ICR-170 preferentially binds to DNA in the beta conformation; factors that increase the temporal stability of this structure, such as adjacent stem-and-loop formation, increase the frequency of ICR-170 binding; (ii) the observed mutagen specificity reflects formation of a preferred ICR-170 intercalative geometry at [sequence in text] sites; (iii) during replication or repair, ICR-170 remains associated with the single-stranded template; (iv) stuttering or strand slippage by the polymerization complex as it encounters the mutagen results in nucleotide duplication; (v) subsequent replication or mismatch repair fixes the insertion into the genome. This mechanism accounts for both the IRC-170 mutagenic specificity and the molecular basis of the highly mutable sites in S. cerevisiae.

Genetic fine structure analysis of the myosin heavy chain gene unc-54 of Caenorhabditis elegans

Taking advantage of the relative ease of detecting single wild-type animals among large numbers of Unc-54 mutant animals, rare intragenic recombinants were recovered between mutations of unc-54 I, the gene coding for the myosin heavy chain of Caenorhabditis elegans body wall musculature. Appropriate closely linked marker mutations were used to improve screening efficiency, to distinguish recombination and conversion events, and to establish the relative order of unc-54 mutations in the gene. From the analysis of more than 400 exceptional chromosomes recovered from tests involving 91 heteroallelic strains, 27 unc-54 mutations have been separated into ten distinct sites. Null mutations, comprising the majority of the mutations studied, are scattered throughout the map, with the e1300 and st60 mutations located at the left and right ends, respectively. Three small deficiencies are clustered at an internal site nearer the left end. Two missense mutations lie to the right of these deficiencies. By correlating these results with molecular investigations of specific mutations, we conclude that the 5′ end of the coding sequence is at the right end of the gene and is located distally on the chromosome. These correlations also demonstrate that the map includes at least 80% of the structural sequence. The genetic map allows us to infer the approximate location of mutations in the coding sequence, and should aid in the genetic dissection of myosin structure and function.

Deletions of a tyrosine tRNA gene in S. cerevisiae

Genetic fine structure analysis of a tyrosine tRNA in yeast revealed that complete deletions of the gene occurred at an unusually high frequency. Among 56 spontaneous mutations at the SUP4 locus, 16 were classified as deletions as judged by their failure to recombine with any other mutations known to map within the gene. Physical analysis of each deletion confirmed the genetic result. The deletions fall into two size classes: ten are 2100 bp deletions and six are 2800 bp deletions. These results imply that the physical structure of the region surrounding the SUP4 locus, which is known to contain short repeated segments, has a direct role in promoting deletions.

GENETIC ORGANIZATION IN CAENORHABDITIS ELEGANS: FINE-STRUCTURE ANALYSIS OF THE unc-22 GENE

Fine-structure analysis of the unc-22 gene of Caenorhabditis elegans has revealed a number of sites that are separable by recombination. Eight new ethyl methanesulfonate-induced recessive mutations of the unc-22 gene have been isolated. Using these new alleles, as well as e66, a number of separable sites have been identified and positioned relative to one another. The map distances obtained are found to be comparable to those associated with intragenic recombination in Drosophila melanogaster, indicating that genetic fine-structure analysis is feasible in Caenorhabditis elegans. Evidence of possible gene conversion is presented. A preliminary estimate of the unc-22 gene size is 2.4 x 10(-2) map units.

THE CAN1 LOCUS OF SACCHAROMYCES CEREVISIAE: FINE-STRUCTURE ANALYSIS AND FORWARD MUTATION RATES

A system of strains and growth media was developed to allow efficient detection of forward mutation, reversion, complementation, and suppression at the canavanine-resistance (CAN1) locus of Saccharomyces cerevisiae. Genetic fine-structure analysis revealed that the map length is at least 40, and possibly as much as 60 X-ray map units; this is the longest gene map yet reported in S. cerevisiae. Allelic complementation was not observed, despite testing of a large number of allele pairs, and alleles suppressible by the ochre suppressor SUP11 were absent from a sample of 48 spontaneous mutants and occurred infrequently (7%) among a sample of ultraviolet-induced mutants. Infrequent mutant types included canavanine-resistant mutants capable of arginine uptake and alleles thought to represent deletions or inversions. In contrast to previous reports in the literature, the spontaneous forward mutation rate at CAN1 did not increase during meiosis.

The Resolving Power of Genetic Fine Structure Analysis in Higher Organisms as Exemplified by Drosophila

Genetic fine structure analysis of a single cistron in Drosophila melanogaster is presented. Large scale sampling, approximating that used in microbial studies, is made possible by the use of a selective scheme generally applicable to all genetic systems in higher organisms. Utilizing this system, it is demonstrated (1) that the mechanism of recombination in higher organisms does indeed permit recombination within a cistron, and (2) that the resolving power of genetic analysis by recombination approaches that of microbial systems.

Physiological Aspects of Bacteriophage Genetics

This chapter discusses bacteriophages as biochemical and genetic systems. The experimental evidence discussed in this chapter leads to the conclusion that two complementary activities take place in the reproduction of bacteriophage,. The first is the replication of the genetic material to ensure genetic continuity of the virus. The second is the specification by the genome of a multiplicity of functions which are necessary for successful passage of the phage through its growth cycle. Both of these aspects of bacteriophage growth pose related problems about molecular mechanisms: one is ultimately concerned with the mechanism of replication of DNA molecules, the other with the mechanism of determination by DNA molecules of the structural details of proteins. The central problem is to determine the relation between the nucleotide sequence of the DNA of a cistron and the amino acid sequence of the protein that it specifies. The best way to attack this problem is to isolate a protein specified by a cistron for which genetic fine structure analysis is possible.