Evidence of tandem duplication of genes in a merodiploid region of Pneumococcal mutants resistant to sulfonamide.

A Pneumococcal mutant, sulr-c, resistant to sulfonamides, and three transformants bearing associated d or d+ resistance markers have earlier been reported to be unstable and show distinct patterns and frequencies of segregating stable progeny lacking the c marker. Each of the four strains showed a characteristic dosage of the genes involved in the merodiploidy. Complementary strands of DNA's from these stable and unstable strains were resolved and homoduplex and heteroduplex hybrids made from the separated DNA strands were used as donors in genetic transformations. Activities of a normal marker (streptomycin resistance) and those involved in the heterozygosity (c, d and d+) were quantitatively measured. From those heteroduplexes made up of opposite strands derived from a heterozygote and a stable strain, the normal marker is transferred efficiently, but the heterozygous markers are not. On the other hand, if both strands of a heteroduplex are derived from different heterozygotic strains, all markers can be transferred with usual efficiency to a stable recipient strain. The lowered efficiency in the former type of heteroduplex is attributed to an inhomology resulting from a tandem duplication in the merodiploid strains, and a postulated DNA repair process stimulated by it while in the form of the donor duplex. The inhomology probably includes (a) a microheterogeneity between the c site and the wild type locus, and (b) a more extensive incompatibility attributable to an extra segment of genome in a tandem duplication covering the c and d sites. The first of these inhomologies produces a lowered efficiency of transfer from all configurations of the particular d allele associated with the mutant c marker, and therefore accounts for the characteristic transfer patterns even from the native merodiploid DNA's.

A study of Pneumococcal merodiploids at the molecular level.

The DNA of a sulfonamide-resistant Pneumococcal strain (heterozygous for sulr-c) and that of three highly resistant and persistently heterozygous cd transformants, derived by introducing sulr-c marker into a stable sulfonamide resistant strain (sulr-d), were studied to analyze the genetic basis of their merodiploidy. The physical properties of the native and denatured DNA from the heterozygotes and the nonheterozygous strains were not distinguishable. The denaturability and the renaturability of biological activity for the heterozygous markers were essentially identical to those of the normal markers. The heterozygosity extends to the closely linked locus giving rise to four different configurations of cd and cd+ transformants, characterized by their frequencies of segregation and donor-marker activities. The marker-activity ratios and the frequency of co-transfer of heterozygous markers were found to remain the same in each when the donor DNA was native, denatured or reannealed without fractionation or reannealed after remixing of resolved strands. Possible models were weighed against these observations and these considerations led to the suggestion that tandem duplication of a gene region may be responsible for the heterozygosity and instability of this region. A more detailed examination of this model will be presented in an accompanying paper.

Chromosomal basis of the merozygosity in a partially deploid mutant of Pneumococcus.

A sulfonamide-resistant mutant of pneumococcus, sulr-c, displays a genetic instability, regularly segregating to wild type. DNA extracts of derivatives of the strain possess transforming activities for both the mutant and wild-type alleles, establishing that the strain is a partial diploid. The linkage of sulr-c to strr-61, a stable chromosomal marker, was established, thus defining a chromosomal locus for sulr-c. DNA isolated from sulr-c cells transforms two mutant recipient strains at the same low efficiency as it does a wild-type recipient, although the mutant property of these strains makes them capable of integrating classical "low-efficiency" donor markers equally as efficiently as "high efficiency" markers. Hence sulr-c must have a different basis for its low efficiency than do classical low efficiency point mutations. We suggest that the DNA in the region of the sulr-c mutation has a structural abnormality which leads both to its frequent segregation during growth and its difficulty in efficiently mediating genetic transformation.

Recombination as a requirement for segregation of a partially diploid mutant of Pneumococcus.

Conditions are described in which the pneumococcal mutant strain sulr-c, resistant to the drug sulfanilamide, gives rise to sensitive segregants resistant to nitrobenzoic acid at a frequency constant with time. This segregant frequency is markedly enhanced upon exposure of the cells to doses of ultraviolet light or mitomycin C that permit survival of 50% to 90% of the cells. Treatment with acridine orange diminishes the segregant frequency. From the known influences of these three agents on genetic recombination, we propose that a recombination event is necessary in the generation of segregants.--During a period of incubation following treatment with ultraviolet light or mitomycin C, cell division resumes and the original segregant frequency is restored. Thus potential segregants are either unable to replicate in the absence of selection, or they are under-represented among the cells dividing soon after treatment.--If the sulr-c mutation is introduced into a mutant pneumococcal strain lacking an ATP-dependent exonuclease activity and deficient in recombination with transforming DNA, segregant frequencies are unaffected. This fact may indicate limits upon the type of recombination event responsible for segregation.

A comparison of two-point, three-point and deletion mapping in the C cistron of rhizobiophage 16-3, with an explanation for the recombination pattern.

A general tendency for additivity prevailed in recombination frequencies for two-point fine-structure mapping of 14 mutants in the C cistron of Rhizobium meliloti phage 16-3, with little evidence of any marker effect. Intracistronic three-point mapping indicated that double crossovers are rare. Deletion mapping indicated that the two- and three-point mapping data gave the correct order of the mutations. A high frequency (5 to 8%) of c/c(+) heterozygotic phage progenies was observed in standard crosses. This pattern implies formation of a relatively long region of heterozygosity. Together with the results of the three-point tests, it suggests certain properties of the branch migration and resolution steps envisioned in current mechanisms of recombination.

Mankind, its numbers, health, and family arrangements: group report 3.

PIP: The population problem is an imbalance between numbers of people and limited resources. The resources of food, energy, and living space have been well documented as finite in a world with population expanding geometrically. Terrorism, breakdown of social order, and other social symptoms may also stem from this overcrowding. The economically more affluent nations are attempting to deal with unregulated fertility by developing and spreading information on means of conception control and granting equality of status to women to compensate for the reduction in the childbearing role. Social systems are very complex and changing the desire for children is quite difficult. In addition, people have a great capacity for ignoring potential disaster when surrounded by it. Liberty of the individual and control must be kept in balance. It is recommended that more effort be made to understand the motivations behind human reproduction and what social incentives exist to reduce family size. There is also need for contraceptive technology and for more effective dissemination thereof. Values and attitudes need to be changed and the mechanisms of communication, the institutional and organizational structures operating in the field of reproductive behavior, and the role of policy determination by the nation need to be studied. There is need for fertility control in conjunction with mortality control. The committee believes that basic minimum components of the quality of life may be imposed upon a people if necessary in order to preserve the diversity of cultural and individual differences.^ieng

Wheelchair rider injuries: causes and consequences for wheelchair design and selection.

An understanding of adverse incidents and injuries sustained by active wheelchair riders, who live and work in the mainstream of society, is needed to improve safety via wheelchair design, selection, and configuration. We interviewed 109 riders who had experienced incidents, in order to identify the causes of incidents and injuries they suffered. Participants reported n = 253 incidents (53% in powered wheelchairs, 47% manual) occurring within a 5-year period, comprised of 106 (42%) "Tips and Falls," 84 (33%) "Component Failures," and 63 (25%) "Other" events. Sixty-eight (27%) of the incidents caused injuries requiring medical attention, including 13 hospitalizations. Direction of Tips and Falls was associated with wheelchair type (manual or powered) and with different riding surfaces. Aspects of wheelchair stability, particularly the effects of wheelchair configuration and of different riding surfaces, are important engineering issues affecting wheelchair safety. Interpretation of the results highlights wheelchair stability mechanics. Potential design improvements are discussed.

Parameters governing bacterial regeneration and genetic recombination after fusion of Bacillus subtilis protoplasts.

Bacterial protoplast fusion, induced by polyethylene glycol, has been made more regular and convenient by further specification and improvement of various steps in the previously used procedure. These have made it possible to obtain regularly 100% regeneration of Bacillus subtilis cells from protoplasts before treatment with polyethylene glycol and yields of 10 to 75% from polyethylene glycol-treated protoplasts. Genetic recombination frequencies do not increase correspondingly. Also, when regeneration is reduced by various experimental conditions, recombination does not decrease in proportion. It is concluded that regeneration of recombinant-forming cells is independently determined and not closely related to the average regeneration for the population. Kinetic studies with varying individual parental or total protoplast concentrations strongly indicate that protoplast collision and contact is not the limiting factor determining the number of genetic recombinants obtained. Recombination approximates a linear, rather than quadratic, function of the total or of the majority protoplast population present, from which it is concluded that fusion events are always adequate to produce substantially more potential recombinants than are registered. The strong effect of the majority/minority ratio upon the number of minority cells that become recombinant is independent of which parent is in excess. This shows in a direct and physiological way that both parents are equivalent partners in their genetic contributions.

A modifier mutation affecting utilization of mannitol in pneumococcus.

The altered growth of a pneumococcal mutant containing marker M, which confers ability to utilize mannitol, and a modifier gene is reported. The modifier gene is closely linked to the M gene and imposes a requirement for growth in 0.4% glucose before growth in mannitol medium. The tentative position of the M gene of pneumococcus is ery-r str-r M sul-rd.

Biparental products of bacterial protoplast fusion showing unequal parental chromosome expression.

Efficiently regenerated single colonies from mixed multiply auxotrophic Bacillus subtilis protoplasts, fused with polyethylene glycol, reveal colonies carrying each of the parent types (biparentals) and recombinant colonies. The latter appear in high yields (up to 1% of certain recombinant classes); the yield of biparentals may be as large as 10%, in the range of the indicated physical fusion events. Many of the biparentals are diploids although, contrary to expectation, they are not complementing prototrophs, but show precisely the phenotype of one (either one) of the parent strains. Extensive pedigree analysis and subcloning of diploid lines show that they can propagate with varying stability on the appropriate parental selective medium to reproduce diploid progeny, parental segregants, and late-appearing recombinants, including some prototrophs. Thus, the principal product of intertype protoplast fusion is a diploid carrying two chromosomes, only one of which is expressed in each particular clone. The extinction of one parental genome is especially well demonstrated when it includes suppression of a normally dominant antibiotic sensitivity marker. In transformation experiments, DNA made from a selected diploid clone was able to transfer several of the unexpressed genes. The structural or topological character of DNA associated with the chromosome extinction remains unexplained.

Chromosome interactions and expression in fused Bacillus protoplasts.

Heterodiploid bacteria can be regenerated from fused mixed protoplasts. In both B. megaterium and B. subtilis, early selective effects during regeneration may strongly affect the phenotype of the products. Among the products are diploid prototrophs, whose stability is still in doubt. It is not known whether the prototrophy results from complementation or from recombination during the selection. In the case of B. subtilis, regeneration in a permissive, uncrowded and non-selective environment leads to production of rather large yields of heterodiploids, either biparental or recombinant. These seem to be only partially stable, but while remaining diploid they give rise to a wide variety of genetic recombinants. In general rather few prototrophs are found when selection is applied to the bacterial diploids. The phenotypic properties of B. subtilis diploids reveal incomplete expression of the demonstrable gene inventory. Biparental diploids appear commonly to show the phenotype of only one of the contained parent types, as if one chromosome remains unexpressed. For diploid recombinants data are insufficient to reveal the status of the chromosomes. Non-expression is believed to be due to structural complexity of particular chromosomes, or their parts. Recombination occurring within bacterial heterodiploid clones appears to satisfy many of the expectations for genetic recombination in eukaryotes--including that of being in part at least classically reciprocal, with intraclonal reciprocals in equivalent numbers. While short map intervals show reduced recombination, large numbers of recombinations (one to two thirds) involve exchanges at or near the terminus and origin of bidirectional replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Reciprocal and nonreciprocal recombination in diploid clones from Bacillus subtilis protoplast fusion: Association with the replication origin and terminus.

The primary heterodiploid bacteria regenerated after Bacillus subtilis fusion, although generally noncomplementing diploids, behave in pedigree analysis as multipotential systems. Individual diploid colonies yielding complete reciprocal recombinant (RR) progeny-often accompanied by one or both parents-constitute 10-30% of the total recombinant-forming units. The RR (reciprocal for 8-11 genes) usually occur in equivalent numbers both among and within individual colonies. Novel for bacteria, they demonstrate that entire parental genomes brought together within a diploid protoplast are retained as two independent replicons able to undergo classical recombination characteristic of eukaryotic gametogenesis. Parental or recombinant genomes are also subject to multiple rounds of recombination without obligate segregation and often not reciprocal. Diploid recombinant clones, sharing streptomycin resistance but reciprocal for auxotrophic markers, have displayed a partial ability to make a facultative shift in chromosome expression. They have also produced two types of prototrophs: a stable one (presumably haploid and recombinant) and an unstable one, (diploid and temporarily complementing at low frequency). It follows that chromosome extinction may affect both parental and recombinant chromosomes and does not interfere with recombination. Analysis of the number and chromosomal distribution of crossovers in all recombinants and those from single diploid clones shows increased frequency of exchange in the regions of the replication origin and terminus, possibly a result of the association of these sites with the cell wall or membrane.