Gene replacement in mycobacteria by using incompatible plasmids

A simple and efficient delivery system was developed for making targeted gene knockouts in Mycobacterium smegmatis. This delivery system relies on the use of a pair of replicating plasmids, which are incompatible. Incompatible plasmids share elements of the same replication machinery and so compete with each other during both replication and partitioning into daughter cells. Such plasmids can be maintained together in the presence of antibiotics; however, removal of selection leads to the loss of one or both plasmids. For mutagenesis, two replicating plasmids based on pAL5000 are introduced; one of these plasmids carries a mutated allele of the targeted gene. Homologous recombination is allowed to take place, and either one or both of the vectors are lost through the pressure of incompatibility, allowing the phenotypic effects of the mutant to be studied. Several different plasmid combinations were tested to optimize loss in the absence of antibiotic selection. pAL5000 carries two replication genes (repA and repB), which act in trans, and the use of vectors that each lack one rep gene and complement each other resulted in the loss of both plasmids in M. smegmatis and Mycobacterium bovis BCG. The rate of loss was increased by the incorporation of an additional incompatibility region in one of the plasmids. To facilitate cloning when the system was used, we constructed plasmid vector pairs that allow simple addition of selection and screening genes on flexible gene cassettes. Using this system, we demonstrated that M. smegmatis pyrF mutants could be isolated at high frequency. This method should also be useful in other species in which pAL5000 replicates, including Mycobacterium tuberculosis.     

Maternal effects and the potential for evolution in a natural population of animals

Maternal effects are widespread and can have dramatic influences on evolutionary dynamics, but their genetic basis has been measured rarely in natural populations. We used cross-fostering techniques and a long-term study of a natural population of red squirrels, Tamiasciurus hudsonicus, to estimate both direct (heritability) and indirect (maternal) influences on the potential for evolution. Juvenile growth in both body mass and size had significant amounts of genetic variation (mass h(2) = 0.10; size h(2) = 0.33), but experienced large, heritable maternal effects. Growth in body mass also had a large positive covariance between direct and maternal genetic effects. The consideration of these indirect genetic effects revealed a greater than three-fold increase in the potential for evolution of growth in body mass (h(2)t = 0.36) relative to that predicted by heritability alone. Simple heritabilities, therefore, may severely underestimate or overestimate the potential for evolution in natural populations of animals.     

Cytotoxic T Cells from Human Immunodeficiency Virus Type 2-Infected Patients Frequently Cross-React with Different Human Immunodeficiency Virus Type 1 Clades

Knowledge of immune mechanisms responsible for the cross-protection between highly divergent viruses such as human immunodeficiency virus type 1 (HIV-1) and HIV-2 may contribute to an understanding of whether virus variability may be overcome in the design of vaccine candidates which are broadly protective across the HIV subtypes. We demonstrate that despite the significant difference in virus amino acid sequence, the majority of HIV-2-infected individuals with different HLA molecules possess a dominant cytotoxic T-cell response which is able to recognize HIV-1 Gag protein. Furthermore, HLA-B5801-positive subjects show broad cross-recognition of HIV-1 subtypes since they mounted a T-cell response that tolerated extensive amino acid substitutions within HLA-B5801-restricted HIV-1 and HIV-2 epitopes. These results suggests that HLA-B5801-positive HIV-2-infected individuals have an enhanced ability to react with HIV-1 that could play a role in cross-protection.Human immunodeficiency virus type 1 (HIV-1) and HIV-2 are related human retroviruses that show various biological and structural differences. HIV-2 is found mainly in West Africa, whereas HIV-1 is spreading throughout the world. HIV-2 is less transmissible, and HIV-2-positive patients exhibit longer clinical latency periods than individuals infected with HIV-1 (23). A recent report has also shown that the mortality in HIV-2-infected individuals is only twice as high as in the uninfected population and, in the majority of adults, survival is not affected by HIV-2 status (31).Although the two viruses are similar in genomic organization, various genetic and enzymatic differences have been found at many stages of the retroviral life cycle. They differ significantly in terms of amino acid sequence, the more conserved being the Pol and Gag sequences, which exhibit less than 60% homology (17).Despite these differences, epidemiological data and animal studies have shown some evidence of cross-protection between the two viral infections. Travers et al. reported that HIV-2-infected women had a lower incidence of HIV-1 infection than did HIV-seronegative women in a cohort of commercial sexual workers in Dakar (37), and rhesus macaques immunized with a recombinant HIV-1 poxvirus vaccine are protected against HIV-2 challenge (2). These studies, though not conclusive (1, 6), suggest that differences in the virus may not necessarily preclude the development of defensive immunity to a subsequent pathogenic infection, an old-fashioned concept pioneered by Jenner, who used cowpox to vaccinate against human smallpox.The immunological basis of cross-protection is largely unknown, and a clear understanding of the role played by the humoral or cell-mediated immune response in HIV protection is still lacking. However, mounting evidence suggests that cytotoxic T-lymphocyte (CTL) response could be the key element. Indeed, the protection afforded in animal models against simian (13) and feline (12) immunodeficiency virus infections is closely correlated with the induction of specific CTL response, and HIV-1 and HIV-2 HLA-B35-restricted cross-reactive CTLs have been postulated to confer protection against repeated HIV exposure (33).CTLs recognize short viral peptides, 8 to 11 amino acids long, that are generated by the intracellular processing of endogenously synthesized viral antigens within the infected cells, which are expressed at the cell surface in the binding groove of HLA class I molecules. The specificity of the T-cell response is determined by the interaction of the antigen-specific T-cell receptor (TCR) with the peptide-HLA complex, and this interaction, together with non-antigen-specific signals, activates the CTLs (15).The presence of cross-reactive CTLs able to lyse HIV-1- or HIV-2-infected cells should be dependent on the extent of conservation between the two viruses within the epitopes selected by particular HLA class I molecules. It is well known that amino acid substitutions within the epitopes can abrogate the CTL response by inhibiting either HLA binding or TCR recognition (32). However, a number of recent studies have shown that T cells can recognize apparently unrelated peptides (10, 41), and crystallographic data have shown physical limits to the TCR epitope specificity due to the limited size of contact between the TCR and the peptide (14), suggesting a flexibility in T-cell recognition of antigen (19).Some individuals with a particular HLA profile which is responsible for presentation of the viral antigen and for selection of the T-cell repertoire may possess a CTL response not affected by mutations within the epitope, as has been demonstrated in subjects with HLA alleles B27 (28) and B35 (33). In these cases, amino acid substitutions within the HIV-1 and -2 epitopes were tolerated by the CTLs.In this study, we have investigated the extent of cross-reacting CTLs between HIV-2 and HIV-1 in a group of HIV-2-infected subjects with different HLA class I types. We have shown that despite differences in amino acid sequence between the two viruses, the majority of HIV-2-positive subjects possess CTLs which are able to recognize HIV-1 Gag protein.Furthermore, analysis of HLA profiles and the fine specificity of the cytotoxic response demonstrated that HLA-B5801-positive subjects show broad cross-recognition of HIV-1 isolates. These subjects mounted a CTL response that tolerated extensive amino acid substitutions within an HLA-B5801-restricted HIV-1 epitope.     

A consideration of the effects of added solutes on the activity of bovine superoxide dismutase.

Pulse radiolysis was used to study the effects of changing the concentration of buffer and added salts on the activity of bovine superoxide dismutase. The results are discussed with reference to other work and are interpreted mainly in terms of ionic-strength effects. The effects are best considered as empirical parameters, which are essential for comparative activity measurements. The specific activity of the enzyme was higher than found in previous pulse-radiolysis studies.     

Post‐weaning parental care increases fitness but is not heritable in North American red squirrels

Most empirical attempts to explain the evolution of parental care have focused on its costs and benefits (i.e. fitness consequences). In contrast, few investigations have been made of the other necessary prerequisite for evolutionary change, inheritance. Here, we examine the fitness consequences and heritability (h²) of a post‐weaning parental care behaviour (territory bequeathal) in a wild population of North American red squirrels. Each year, a subset (average across all years = 19%) of reproductive females bequeathed their territory to a dependent offspring. Bequeathing females experienced higher annual reproductive success and did not suffer a survival cost to themselves relative to those females retaining their territory. Bequeathing females thus realized higher relative annual fitness [ω = 1.18 ± 0.03 (SE)] than nonbequeathing females [ω = 0.96 ± 0.02 (SE)]. Additive genetic influences on bequeathal behaviour, however, were not significantly different from 0 (h² = 1.9 × 10^?3; 95% highest posterior density interval = 3.04 × 10^?8 to 0.37) and, in fact, bequeathal behaviour was not significantly repeatable (R = 2.0 × 10^?3; 95% HPD interval =0–0.27). In contrast, directional environmental influences were apparent. Females were more likely to bequeath in years following low food abundance and when food availability in the upcoming autumn was high. Despite an evident fitness benefit, a lack of heritable genetic variance will constrain evolution of this trait.     

The nature of nurture in a wild mammal's fitness

Genetic variation in fitness is required for the adaptive evolution of any trait but natural selection is thought to erode genetic variance in fitness. This paradox has motivated the search for mechanisms that might maintain a population''s adaptive potential. Mothers make many contributions to the attributes of their developing offspring and these maternal effects can influence responses to natural selection if maternal effects are themselves heritable. Maternal genetic effects (MGEs) on fitness might, therefore, represent an underappreciated source of adaptive potential in wild populations. Here we used two decades of data from a pedigreed wild population of North American red squirrels to show that MGEs on offspring fitness increased the population''s evolvability by over two orders of magnitude relative to expectations from direct genetic effects alone. MGEs are predicted to maintain more variation than direct genetic effects in the face of selection, but we also found evidence of maternal effect trade-offs. Mothers that raised high-fitness offspring in one environment raised low-fitness offspring in another environment. Such a fitness trade-off is expected to maintain maternal genetic variation in fitness, which provided additional capacity for adaptive evolution beyond that provided by direct genetic effects on fitness.