Enterotoxin B formation by fermentation mutants of Staphylococcus aureus.

An appreciable fraction of carbohydrate-negative (car) mutants of Staphylococcus aureus strains ATCC 14458, 778, and S-6 exhibit increased enterotoxin B (SEB) production. In addition, some lac and mtl mutants of these strains also display enhanced SEB formation. All such mutants appear to be point mutations. Mutagen-induced reversions of high SEB producing car, mtl, or lac mutants yield varying amounts of SEB and some clones seem to be restored to the characteristics of the parent type. A few sequentially isolated lac, mtl double mutants of strain 778 elaborate much more or much less SEB than either the lac or the mtl single mutants.     

Membrane mutations and production of enterotoxin B and alpha hemolysin in Staphylococcus aureus.

Staphylococcus aureus strain S-6, which produces enterotoxin type B (SEB), and strain 10-275, a high toxin-producing mutant derived from S-6, display pronounced differences in dye sensitivity, osmotic stability, and bacitracin sensitivity. Such characteristics are consistent with the concept that strain 10-275 is a membrane mutant of strain S-6. Some membrane mutants of S. aureus strain 14458 exhibit about two- to three-fold increases in SEB production whereas other membrane mutants show about twofold increases in alpha-hemolysin production. It is suggested that specific and independent membrane mutations control the secretory processes resulting in the extracellular elaboration of these exoproteins.     

Increased outer membrane resistance to ethylenediaminetetraacetate and cations in novel lipid A mutants.

Polymyxin-resistant pmrA mutants of Salmonella typhimurium differed from their parents in that they were resistant to tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-lysozyme, tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-deoxycholate, and tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-bacitracin. Tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate released about 50% less lipopolysaccharide from the pmrA strains than from the parental strains when the bacteria were grown in L-broth containing 2 mM Ca2+. Protamine, polylysine, octapeptin, benzalkonium chloride, cold NaCl, cold MgCl2, or cold tris(hydroxymethyl)aminomethane hydrochloride (pH 7.2) caused no leakage or markedly less leakage of periplasmic beta-lactamase from a pmrA mutant than from its parent strain. pmrA mutants were more resistant than their parent strains to protamine and polylysine but not to octapeptin or benzalkonium chloride, as measured by the ability of these agents to kill the bacteria or to sensitize them to deoxycholate-induced lysis. The pmrA strains did not differ from their parent strains in sensitivity to several antibiotics, in porin function (as measured by cephaloridine diffusion across the outer membrane), or in outer membrane-associated phospholipase A activity.     

Mutations affecting glutamine synthetase activity in Salmonella typhimurium.

A positive selection procedure has been devised for isolating mutant strains of Salmonella typhimurium with altered glutamine synthetase activity. Mutants are derived from a histidine auxotroph by selecting for ability to grow on D-histidine as the sole histidine source. We hypothesize that the phenotype may be based on a regulatory increase in the activities of the D-histidine racemizing enzymes, but this has not been established. Spontaneous glutamine-requiring mutants isolated by the above selection procedure have two types of alterations in glutamine synthetase activity. Some have less than 10% of parent activity. Others have significant glutamine synthetase activity, but the enzyme have an altered response to divalent cations. Activity in mutants of the second type mimics that of highly adenylylated wild-type enzyme, which is believed to be in-active in vivo. Glutamine synthetase from one such mutant is more heat labile than wild-type enzyme, indicating that it is structurally altered. Mutations in all strains are probably in the glutamine synthetase structural gene (glnA). They are closely linked on the Salmonella chromosome and lie at about min 125. The mutants have normal glutamate dehydrogenase activity.     

Mutant strains of Rhodopseudomonas spheroides which form photosynthetic pigments aerobically in the dark. Growth characteristics and enzymic activities

Mutant strains of Rhodopseudomonas spheroides have been isolated which contain 5–50 times more bacteriochlorophyll and carotenoids than the wild type when grown under highly aerobic conditions in the dark. Their pigment content is similar to the wild type when grown in the light. One of the mutants (TA-R) grew more slowly than its parent strain under aerobic conditions but formed pigments at about 60% of the rate observed under photosynthetic conditions. The other mutants grew at rates similar to the wild type under all conditions. Synthesis of bacteriochlorophyll by suspensions of the mutants began without delay upon transfer from conditions of high to low aeration. In contrast to the wild type, magnesium protoporphyrin-S-adenosylmethionine methyltransferase (EC 2.1.1.11) activity in particulate preparations from the mutants was not repressed by growth under aerobic conditions in the light or dark. Ribulose diphosphate carboxylase (EC 4.1.1.39) activity was repressed by O₂ in the mutants as in the wild type. Other enzyme activities were compared in mutant TA-R and its parent strain grown under the same conditions. NADH oxidase activity in particles from aerobically grown TA-R was about one third that found in the parent strain. However, the respiration rates of the intact cells did not differ. Light inhibited the respiration of aerobically grown TA-R, indicating that the bacteriochlorophyll formed under these conditions had photochemical activity. It is concluded that the insensitivity of the mutants to O₂ repression is due to defects in the regulatory system which controls formation of the enzymes concerned in pigment synthesis.     

In vitro and in vivo characterization of alkyl hydroperoxide reductase mutant strains of Helicobacter hepaticus

Mutant strains in the tsaA gene encoding alkyl hydroperoxide reductase were more sensitive to O(2) and to oxidizing agents (paraquat, cumene hydroperoxide and t-butylhydroperoxide) than the wild type, but were markedly more resistant to hydrogen peroxide. The mutant strains resistance phenotype could be attributed to a 4-fold and 3-fold increase in the catalase protein amount and activity, respectively compared to the parent strain. The wild type did not show an increase in catalase expression in response to sequential increases in O(2) exposure or to oxidative stress reagents, so an adaptive compensatory mutation has probably occurred in the mutants. In support of this, chromosomal complementation of tsaA mutants restored alkyl hydroperoxide reductase, but catalase was still up-expressed in all complemented strains. The katA promoter sequence was the same in all mutant strains and the wild type. Like its Helicobacter pylori counterpart strain, a H. hepaticus tsaA mutant contained more lipid hydroperoxides than the wild type strain. Hepatic tissue from mice inoculated with a tsaA mutant had lesions similar to those inoculated with the wild type, and included coagulative necrosis of hepatocytes. The liver and cecum colonizing abilities of the wild type and tsaA mutant were comparable. Up-expression of catalase in the tsaA mutants likely permits the bacterium to compensate (in colonization and virulence attributes) for the loss of an otherwise important oxidative stress-combating enzyme, alkyl hydroperoxide reductase. The use of erythromycin resistance insertion as a facile way to screen for gene-targeted mutants, and the chromosomal complementation of those mutants are new genetic procedures for studying H. hepaticus.     

Screening and selection of exopolysaccharide-producing strains of Lactobacillus delbrueckii subsp. bulgaricus

AIMS: The selection of exopolysaccharide (EPS)-producing strains of Lactobacillus delbrueckii subsp. bulgaricus. METHODS AND RESULTS: Improved EPS-overproducing strains of L. delbrueckii subsp. bulgaricus were derived by chemical mutagenesis and selection. Initial screening of the chemically induced mutant pool relied primarily on the selection of strains with raised levels of lactic acid and reduced biomass formation. Supporting selection criteria used were ropiness and colonial mucoidy. Final screening of candidate strains undertaken in a semi-defined medium in batch culture, resulted in the selection of a mutant with a 35% improvement in specific EPS yield relative to the parent strain. CONCLUSIONS: Initial selection of mutants of L. delbrueckii subsp. bulgaricus on the basis of enhanced formation of lactate and reduced biomass formation, coupled with a ropy or mucoid phenotype, proved to be a satisfactory means of isolating strains with the potential for a higher level of specific EPS production than the parent strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay protocol allowed for the selection of an EPS-overproducing strain of L. delbrueckii subsp. bulgaricus. Such strains are useful for the purposes of metabolic studies related to EPS-production.     

Uptake of exogenous coenzyme Q and transport to mitochondria is required for bc1 complex stability in yeast coq mutants.

Coenzyme Q (Q) is an essential component of the mitochondrial respiratory chain in eukaryotic cells but also is present in other cellular membranes where it acts as an antioxidant. Because Q synthesis machinery in Saccharomyces cerevisiae is located in the mitochondria, the intracellular distribution of Q indicates the existence of intracellular Q transport. In this study, the uptake of exogenous Q(6) by yeast and its transport from the plasma membrane to mitochondria was assessed in both wild-type and in Q-less coq7 mutants derived from four distinct laboratory yeast strains. Q(6) supplementation of medium containing ethanol, a non-fermentable carbon source, rescued growth in only two of the four coq7 mutant strains. Following culture in medium containing dextrose, the added Q(6) was detected in the plasma membrane of each of four coq7 mutants tested. This detection of Q(6) in the plasma membrane was corroborated by measuring ascorbate stabilization activity, as catalyzed by NADH-ascorbate free radical reductase, a transmembrane redox activity that provides a functional assay of plasma membrane Q(6). These assays indicate that each of the four coq7 mutant strains assimilate exogenous Q(6) into the plasma membrane. The two coq7 mutant strains rescued by Q(6) supplementation for growth on ethanol contained mitochondrial Q(6) levels similar to wild type. However, the content of Q(6) in mitochondria from the non-rescued strains was only 35 and 8%, respectively, of that present in the corresponding wild-type parental strains. In yeast strains rescued by exogenous Q(6), succinate-cytochrome c reductase activity was partially restored, whereas non-rescued strains contained very low levels of activity. There was a strong correlation between mitochondrial Q(6) content, succinate-cytochrome c reductase activity, and steady state levels of the cytochrome c(1) polypeptide. These studies show that transport of extracellular Q(6) to the mitochondria operates in yeast but is strain-dependent. When Q biosynthesis is disrupted in yeast strains with defects in the intracellular transport of exogenous Q, the bc(1) complex is unstable. These results indicate that delivery of exogenous Q(6) to mitochondria is required fore activity and stability of the bc(1) complex in yeast coq mutants.     

Construction of less neurovirulent polioviruses by introducing deletions into the 5'' noncoding sequence of the genome.

Viral attenuation may be due to lowered efficiency of certain steps essential for viral multiplication. For the construction of less neurovirulent strains of poliovirus in vitro, we introduced deletions into the 5' noncoding sequence (742 nucleotides long) of the genomes of the Mahoney and Sabin 1 strains of poliovirus type 1 by using infectious cDNA clones of the virus strains. Plaque sizes shown by deletion mutants were used as a marker for rate of viral proliferation. Deletion mutants of both the strains thus constructed lacked a genome region of nucleotide positions 564 to 726. The sizes of plaques displayed by these deletion mutants were smaller than those by the respective parental viruses, although a phenotype referring to reproductive capacity at different temperatures (rct) of viruses was not affected by introduction of the deletion. Monkey neurovirulence tests were performed on the deletion mutants. The results clearly indicated that the deletion mutants had much less neurovirulence than with the corresponding parent viruses. Production of infectious particles and virus-specific protein synthesis in cells infected with the deletion mutants started later than in those infected with the parental viruses. The rate at which cytopathic effect progressed was also slower in cells infected with the mutants. Phenotypic stability of the deletion mutant for small-plaque phenotype and temperature sensitivity was investigated after passaging the mutant at an elevated temperature of 37.5 degrees C. Our data strongly suggested that the less neurovirulent phenotype introduced by the deletion is very stable during passaging of the virus.     

NO3– nutrition and salt tolerance in the cyanobacterium Anabaena sp. PCC 7120 and mutant strains

Growth of the cyanobacterium Anabaena sp. PCC 7120 and its nitrate assimilation-defective mutants was inversely proportional to the NaCl concentration in the medium. Presence of nitrate in the saline medium protected the growth of the parent but not of the mutant strains from salt toxicity. On the other hand, ammonium nitrogen protected the growth of all the strains from salt toxicity. However, the effect was less than that of nitrate. An altered sodium transport system was evident in the mutant strains and was most marked in mutant SP9. The cellular sodium concentration in parent and mutant strains also varied. Although mutant SP9 exhibited the lowest level of cellular sodium, it was as sensitive to salt toxicity as other strains. It is assumed that merely the presence of a toxic level of NaCl in the ambient environment is sufficient to damage the structural and functional components of the plasma membrane.     

Selection and properties of phototaxis-deficient mutants of Halobacterium halobium

A method for isolating phototaxis-deficient (Pho-) mutants of Halobacterium halobium was developed. The procedure makes use of a flashing repellent light to induce frequent reversals of swimming direction by responsive cells, thereby impeding their migration along a small capillary and resulting in a spatial separation of the parent population and a population enriched for Pho- cells. Two classes of Pho- mutants were obtained by this selection scheme: those which have lost the chemotactic response (Che-) as well as phototaxis sensitivity (general taxis mutants), and those which are defective in steps specific to phototaxis (photosignaling mutants). In the latter class, several retinal synthesis mutants were isolated, as well as a strain which fit the expected properties of a mutant lacking a functional photoreceptor protein. On the basis of spectroscopic and swimming behavior studies, the retinal-containing protein, slow-cycling or sensory rhodopsin (SR), was previously proposed to be a dual-function sensory receptor mediating both attractant and repellent photosensing. The receptor mutant Pho81 fulfills two predictions which provide direct genetic evidence for this proposal. The mutant has lost SR photoactivity as determined by spectroscopic measurements, and it has simultaneously lost both attractant and repellent phototaxis sensitivity. Comparison of [3H]retinal-labeled membrane proteins from the mutant and its SR-containing parent implicated a 25,000 Mr polypeptide as the chromophoric polypeptide of SR.     

Mutant of Escherichia coli K-12 Deficient for Detergent-Resistant Phospholipase A

A mutant deficient for detergent-resistant (DR) phospholipase A was isolated from Escherichia coli K-12. Because the enzyme is membrane-bound and the substrate is a lipid, a special procedure was developed for isolating mutants deficient for the enzyme from agar plates. A sodium dodecyl sulfate (SDS)-sensitive mutant was used as a parental strain for the isolation of DR phospholipase A-deficient mutant. Soft agar containing an unsaturated fatty acid auxotroph and SDS was poured over colonies of the parental strain. The cells were easily solubilized with SDS, and phospholipids were efficiently digested by DR phospholipase A from the colonies on an agar plate. Fatty acids released supported the growth of the indicator bacteria. After the cells of the parent were mutagenized with nitrosoguanidine, colonies which could not support the growth of an unsaturated fatty acid auxotroph in the presence of SDS were selected. Four mutants were isolated after in vitro scre[UNK]ning of DR phospholipase A activity of 30 halo-less clones. Since an extract of the parent strain mixed with that of a mutant strain was still active, it was concluded that the inability to hydrolyze phospholipids was not due to the accumulation of inhibitory substance; the activity of DR phospholipase A in the mutant was less than 1% of the parental activity. Physiological studies indicated that DR phospholipase A is not essential for the growth of E. coli.     

Biochemical Properties of Haploid and Diploid Strains of Penicillium chrysogenum

An intensive parasexual genetics program in which industrial strains of Penicillium chrysogenum were used culminated in the isolation of a number of heterozygous diploid strains. The diploid clones were selected from heterokaryons formed from matings between mutant strains having complementary biochemical and conidial color markers. Several diploid cultures were compared with their haploid wild-type parents and other distantly related production strains on the basis of a variety of cultural and physiological criteria. The diploid strains characteristically produced conidia of larger volume and higher deoxyribonucleic acid content. Some were vigorous with respect to growth rate and onset and degree of conidiation. One diploid strain (WC-9) had a 46% greater oxygen uptake rate and oxidized glucose at a 57% greater rate than its haploid parent (M-2). It also produced 33% higher concentrations of β-galactosidase, 66% more alkaline protease, and 53% more glucose oxidase than the M-2 haploid parent. The selection of rare stable diploid mold cultures through the use of parasexual genetics offers a unique approach to the direct selection of mutants with potential for increased enzyme formation.     

Characterization of polyagglutinating and surface antigens in Pseudomonas aeruginosa

Mutants of Pseudomonas aeruginosa PAC1R (serotype O:3) which were resistant to bacteriophage D were isolated and shown to react with O:5d, O:9 and O:13 antisera as well as O:3. Antisera to the parent strain and to the three polyagglutinating (PA) mutants also showed cross-reactions. The mutants differed from the parent strain in their lipopolysaccharide (LPS) composition. The LPS from two of the three mutants yielded high molecular weight polysaccharide fractions. Although the high molecular weight fraction from one of the mutants contained the amino sugars and other components characteristic of the O:3 serotype strains, its mobility on Sephadex G75 was different from that of the parent strain. The high molecular weight material from the second mutant lacked the O-antigenic determinants but these were present in a semi-rough LPS fraction. The third mutant appeared rough and completely lacked the O-antigenic components. These three mutants were compared with the parent strain and with a non-agglutinating LPS-defective mutant which lacked both O-antigenic side chains and all neutral sugars in the outer core. Agglutination with absorbed sera and haemagglutination using purified LPS and ELISA detection suggested that wall components other than LPS were responsible for some of the cross-reactions observed. The components responsible were detected after SDS-PAGE of crude outer membrane fractions by a combination of Coomassie blue and silver-staining and antigenic components were detected by immunoelectrophoresis and ELISA-linked immunoblotting of the gels. The main antigenic determinants detected by antiserum to the parent strain were in the high molecular weight O-polysaccharide fractions and in the semirough fractions of the LPS, with some activity due to the H protein of the outer membrane. O:5d antisera reacted with unidentified high molecular weight polysaccharide fractions. Cross-reactions with the O:9 antiserum appeared to be due mainly to the F porin and, to a lesser extent, to the G and E proteins of the outer membrane. O:13 antiserum reacted with high molecular weight polysaccharide fractions but also with the rough core and F and H protein. Cross-reactivity of the other three mutant antisera could largely be interpreted in terms of the outer membrane components exposed in each strain. One reacted strongly with the F porin and the rough core, while the others reacted with a number of protein and LPS-derived fractions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of pure and sectored mutant clones in excision-proficient and deficient strains of yeast.

We have found that UV-induced mutation frequency in a forward non-selective assay system (scoring white adex ade2 double auxotroph mutants among the red pigmented ade2 clones) increases linearly with dose up to a maximum frequency of about 3 X 10(-3) mutants per survivor and then declines in both RAD wild-type and rad2 excision deficient strains of Saccharomyces cerevisiae. Mutation frequencies of the RAD and the rad2 strains plotted against survival are nearly identical over the entire survival range. On this basis we conclude that unexcised pyrimidine dimers are the predominant type of pre-mutational lesions in both strains. In the RAD wild-type strain pure mutant clones outnumber sectors in a 10:1 ratio at all doses used; in rad2 this ratio varies from 1:1 at low doses up to 10:1 at high doses. As others have concluded for wild-type strains we find also in the rad2 strain that pure clone formation cannot be accounted for quantitatively by lethal sectoring events alone. We conclude that heteroduplex repair is a crucial step in pure mutant clone formation and we examine the plausibility of certain macromolecular mechanisms according to which heteroduplex repair may be coupled with replication, repair and sister strand exchange in yeast mutagenesis.     

In vitro and in vivo characterization of alkyl hydroperoxide reductase mutant strains of Helicobacter hepaticus

Mutant strains in the tsaA gene encoding alkyl hydroperoxide reductase were more sensitive to O₂ and to oxidizing agents (paraquat, cumene hydroperoxide and t-butylhydroperoxide) than the wild type, but were markedly more resistant to hydrogen peroxide. The mutant strains resistance phenotype could be attributed to a 4-fold and 3-fold increase in the catalase protein amount and activity, respectively compared to the parent strain. The wild type did not show an increase in catalase expression in response to sequential increases in O₂ exposure or to oxidative stress reagents, so an adaptive compensatory mutation has probably occurred in the mutants. In support of this, chromosomal complementation of tsaA mutants restored alkyl hydroperoxide reductase, but catalase was still up-expressed in all complemented strains. The katA promoter sequence was the same in all mutant strains and the wild type. Like its Helicobacter pylori counterpart strain, a H. hepaticus tsaA mutant contained more lipid hydroperoxides than the wild type strain. Hepatic tissue from mice inoculated with a tsaA mutant had lesions similar to those inoculated with the wild type, and included coagulative necrosis of hepatocytes. The liver and cecum colonizing abilities of the wild type and tsaA mutant were comparable. Up-expression of catalase in the tsaA mutants likely permits the bacterium to compensate (in colonization and virulence attributes) for the loss of an otherwise important oxidative stress-combating enzyme, alkyl hydroperoxide reductase. The use of erythromycin resistance insertion as a facile way to screen for gene-targeted mutants, and the chromosomal complementation of those mutants are new genetic procedures for studying H. hepaticus.     

Forskolin-resistant Y1 mutants harbor defects associated with the guanyl nucleotide-binding regulatory protein, Gs

The properties of the adenylate cyclase from forskolin-resistant mutants of Y1 adrenocortical tumor cells was compared with the properties of the enzyme from parental Y1 cells in order to localize the site of mutation. In parental Y1 cells, forskolin stimulated adenylate cyclase activity with kinetics suggestive of an interaction at two sites; in mutant cells, forskolin resistance was characterized by a decrease in enzymatic activity at both sites. Forskolin potentiated the enzyme's responses to NaF and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) in parent and mutant clones, and the mutant enzyme showed the same requirements for Mg2+ and Mn2+ as did the parent enzyme. The adenylate cyclase associated with forskolin-resistant mutants was insensitive to ACTH and was less responsive to Gpp(NH)p than was the parent enzyme. In parental Y1 cells and in the forskolin-resistant mutants, cholera toxin catalyzed the transfer of [32P]ADP-ribose from [32P]NAD+ into three membrane proteins associated with the alpha subunit of Gs; however, the amount of labeled ADP-ribose incorporated into mutant membranes was reduced by as much as 70%. Both parent and mutant membranes were labeled by pertussis toxin to the same extent. The insensitivity of the mutant adenylate cyclase to ACTH and Gpp(NH)p and the selective resistance of the mutant membranes to cholera toxin-catalyzed ADP-ribosylation suggest that a specific defect associated with Gs is involved in the mutation to forskolin resistance in Y1 cells.     

Comparative pathogenicity of auxotrophic mutants of Candida albicans

An induced mutant of Candida albicans with greatly decreased virulence for mice is described. The mutant was one of five auxotrophic mutants obtained by ultraviolet irradiation of a clinical isolate (strain MY 1044). The five mutants included two methionine auxotrophs, one methionine-cysteine auxotroph, one temperature-sensitive serine auxotroph, and one auxotroph with unknown growth requirements. Each of the mutants produced normal mycelium and had a normal profile of susceptibility to four antifungal drugs. The virulence of each mutant was compared with the parent strain by LD50 determination in mice. Four of the five auxotrophs exhibited LD50's that were not significantly different from the parent strain (mean LD50 = 7.5 x 10(5) cells). However, the temperature-sensitive serine auxotroph was significantly less virulent than the parent strain (LD50 greater than 10(7) cells), even though it grew well in vivo and in mouse serum at 37 degrees C in vitro. Use of this mutant in conjunction with its "isogenic" parent should help to elucidate true virulence factors in C. albicans.     

Inhibition of citric acid accumulation by manganese ions in Aspergillus niger mutants with reduced citrate control of phosphofructokinase.

Mutant strains of Aspergillus niger with reduced citrate control of carbohydrate catabolism (cic mutants) grow faster than the parent strain on media containing 5% (wt/vol) citrate. The mutants tolerated a higher intracellular citrate concentration than the parent strain. One mutant (cic-7/3) contained phosphofructokinase activity significantly less sensitive towards citrate than the enzyme from the parent strain. When this mutant was grown under citrate-accumulating conditions, acidogenesis was far less sensitive to inhibition by Mn2+ than in the parent strain. Some of the cic mutants also showed altered citrate inhibition of NADP-specific isocitrate dehydrogenase.     

Marker Effects in the Genetic Transduction of Tryptophan Mutants of E. COLI

Recombination frequencies have been determined in crosses involving 28 mutant strains for 20 of which the site of the alteration is known from studies of amino-acid substitutions in the protein products. Three of these mutants showed especially high frequencies of recombination when crossed to other single mutants or when crossed to a strain carrying two alterations at opposite ends of the trpA gene. There is no obvious molecular explanation of the high recombination of these three mutants. They include one missense mutant, one amber and one ochre. The low-frequency recombination mutants include all these same classes as well as frameshift mutants. There is nothing unique about the intragenic location of the high-recombination mutants; in each case there is at least one low-recombination mutant in the same codon.-Crosses involving mutants which were isolated in an altered wild type have shown that the behavior of a high-recombination mutant does not result from its molecular configuration alone, but from its combination with the homologous wild-type sequence from the other parent.-Several lines of evidence indicate that recombination in this system frequently involves closely-spaced double exchanges (about 40 codons apart).