Relationship between evolutionary rate and cellular location among the Inv/Spa invasion proteins of Salmonella enterica.

For 21 strains of Salmonella enterica, nucleotide sequences were obtained for three invasion genes, spaO, spaP, and spaQ, of the chromosomal inv/spa complex, the products of which form a protein export system required for entry of the bacteria into nonphagocytic host cells. These genes are present in all eight subspecies of the salmonellae, and homologues occur in a variety of other bacteria, including the enteric pathogens Shigella and Yersinia, in which they are plasmid borne. Evolutionary diversification of the invasion genes among the subspecies of S. enterica has been generally similar in pattern and average rate to that of housekeeping genes. However, the range of variation in evolutionary rate among the invasion genes is unusually large, and there is a relationship between the evolutionary rate and cellular location of the invasion proteins, possibly reflecting diversifying selection on exported proteins in adaptation to variable host factors in extracellular environments. The SpaO protein, which is hypervariable in S. enterica and exhibits only 24% sequence identity with its homologues in Shigella and Yersinia, is secreted. In contrast, the membrane-associated proteins SpaP, SpaQ, and InvA are weakly polymorphic and have > 60% sequence identity with the corresponding proteins of other enteric bacteria. Acquisition of the inv/spa genes may have been a key event in the evolution of the salmonellae as pathogens, following which the invention of flagellar phase shifting facilitated niche expansion to include warm-blooded vertebrates.

Suppression of protein synthesis in brain during hibernation involves inhibition of protein initiation and elongation

Protein synthesis (PS) has been considered essential to sustain mammalian life, yet was found to be virtually arrested for weeks in brain and other organs of the hibernating ground squirrel, Spermophilus tridecemlineatus. PS, in vivo, was below the limit of autoradiographic detection in brain sections and, in brain extracts, was determined to be 0.04% of the average rate from active squirrels. Further, it was reduced 3-fold in cell-free extracts from hibernating brain at 37°C, eliminating hypothermia as the only cause for protein synthesis inhibition (active, 0.47 ± 0.08 pmol/mg protein per min; hibernator, 0.16 ± 0.05 pmol/mg protein per min, P < 0.001). PS suppression involved blocks of initiation and elongation, and its onset coincided with the early transition phase into hibernation. An increased monosome peak with moderate ribosomal disaggregation in polysome profiles and the greatly increased phosphorylation of eIF2α are both consistent with an initiation block in hibernators. The elongation block was demonstrated by a 3-fold increase in ribosomal mean transit times in cell-free extracts from hibernators (active, 2.4 ± 0.7 min; hibernator, 7.1 ± 1.4 min, P < 0.001). No abnormalities of ribosomal function or mRNA levels were detected. These findings implicate suppression of PS as a component of the regulated shutdown of cellular function that permits hibernating ground squirrels to tolerate “trickle” blood flow and reduced substrate and oxygen availability. Further study of the factors that control these phenomena may lead to identification of the molecular mechanisms that regulate this state.

Cytotoxic T lymphocytes and viral turnover in HIV type 1 infection

To understand the role of the immune system in limiting HIV type 1 replication, it is critical to know to what extent the rapid turnover of productively infected cells is caused by viral cytopathicity or by immune-mediated lysis. We show that uncultured peripheral blood mononuclear cells of many patients contain cytotoxic T lymphocytes (CTL) that lyse target cells—at plausible peripheral blood mononuclear cell-to-target ratios—with half-lives of less than 1 day. In 23 patients with CD4 counts ranging from 10 to 900 per μl, the average rate of CTL-mediated lysis corresponds to a target cell half-life of 0.7 day. We develop mathematical models to calculate the turnover rate of infected cells subjected to immune-mediated lysis and viral cytopathicity and to estimate the fraction of cells that are killed by CTL as opposed to virus. The models provide new interpretations of drug treatment dynamics and explain why the observed rate of virus decline is roughly constant for different patients. We conclude that in HIV type 1 infection, CTL-mediated lysis can reduce virus load by limiting virus production, with small effects on the half-life of infected cells.

Long term trends in the evolution of H(3) HA1 human influenza type A

We have studied the HA1 domain of 254 human influenza A(H3N2) virus genes for clues that might help identify characteristics of hemagglutinins (HAs) of circulating strains that are predictive of that strain’s epidemic potential. Our preliminary findings include the following. (i) The most parsimonious tree found requires 1,260 substitutions of which 712 are silent and 548 are replacement substitutions. (ii) The HA1 portion of the HA gene is evolving at a rate of 5.7 nucleotide substitutions/year or 5.7 × 10−3 substitutions/site per year. (iii) The replacement substitutions are distributed randomly across the three positions of the codon when allowance is made for the number of ways each codon can change the encoded amino acid. (iv) The replacement substitutions are not distributed randomly over the branches of the tree, there being 2.2 times more changes per tip branch than for non-tip branches. This result is independent of how the virus was amplified (egg grown or kidney cell grown) prior to sequencing or if sequencing was carried out directly on the original clinical specimen by PCR. (v) These excess changes on the tip branches are probably the result of a bias in the choice of strains to sequence and the detection of deleterious mutations that had not yet been removed by negative selection. (vi) There are six hypervariable codons accumulating replacement substitutions at an average rate that is 7.2 times that of the other varied codons. (vii) The number of variable codons in the trunk branches (the winners of the competitive race against the immune system) is 47 ± 5, significantly fewer than in the twigs (90 ± 7), which in turn is significantly fewer variable codons than in tip branches (175 ± 8). (viii) A minimum of one of every 12 branches has nodes at opposite ends representing viruses that reside on different continents. This is, however, no more than would be expected if one were to randomly reassign the continent of origin of the isolates. (ix) Of 99 codons with at least four mutations, 31 have ratios of non-silent to silent changes with probabilities less than 0.05 of occurring by chance, and 14 of those have probabilities <0.005. These observations strongly support positive Darwinian selection. We suggest that the small number of variable positions along the successful trunk lineage, together with knowledge of the codons that have shown positive selection, may provide clues that permit an improved prediction of which strains will cause epidemics and therefore should be used for vaccine production.

Real time observation of anaphase in vitro.

We used digital fluorescence microscopy to make real-time observations of anaphase chromosome movement and changes in microtubule organization in spindles assembled in Xenopus egg extracts. Anaphase chromosome movement in these extracts resembled that seen in living vertebrate cells. During anaphase chromosomes moved toward the spindle poles (anaphase A) and the majority reached positions very close to the spindle poles. The average rate of chromosome to pole movement (2.4 microns/min) was similar to earlier measurements of poleward microtubule flux during metaphase. An increase in pole-to-pole distance (anaphase B) occurred in some spindles. The polyploidy of the spindles we examined allowed us to observe two novel features of mitosis. First, during anaphase, multiple microtubule organizing centers migrated 40 microns or more away from the spindle poles. Second, in telophase, decondensing chromosomes often moved rapidly (7-23 microns/min) away from the spindle poles toward the centers of these asters. This telophase chromosome movement suggests that the surface of decondensing chromosomes, and by extension those of intact nuclei, bear minus-end-directed microtubule motors. Preventing the inactivation of Cdc2/cyclin B complexes by adding nondegradable cyclin B allowed anaphase A to occur at normal velocities, but reduced the ejection of asters from the spindles, blocked chromosome decondensation, and inhibited telophase chromosome movement. In the presence of nondegradable cyclin B, chromosome movement to the poles converted bipolar spindles into pairs of independent monopolar spindles, demonstrating the role of sister chromatid linkage in maintaining spindle bipolarity.

The organization of seismicity on fault networks.

Although models of homogeneous faults develop seismicity that has a Gutenberg-Richter distribution, this is only a transient state that is followed by events that are strongly influenced by the nature of the boundaries. Models with geometrical inhomogeneities of fracture thresholds can limit the sizes of earthquakes but now favor the characteristic earthquake model for large earthquakes. The character of the seismicity is extremely sensitive to distributions of inhomogeneities, suggesting that statistical rules for large earthquakes in one region may not be applicable to large earthquakes in another region. Model simulations on simple networks of faults with inhomogeneities of threshold develop episodes of lacunarity on all members of the network. There is no validity to the popular assumption that the average rate of slip on individual faults is a constant. Intermediate term precursory activity such as local quiescence and increases in intermediate-magnitude activity at long range are simulated well by the assumption that strong weakening of faults by injection of fluids and weakening of asperities on inhomogeneous models of fault networks is the dominant process; the heat flow paradox, the orientation of the stress field, and the low average stress drop in some earthquakes are understood in terms of the asperity model of inhomogeneous faulting.

Immunoglobulin variable region hypermutation in hybrids derived from a pre-B- and a myeloma cell line.

Somatic mutation of the variable (V) regions of immunoglobulin genes occurs in vivo at rates that have been estimated to be between 10(-3) and 10(-4) per bp per generation. To study this process in vitro, the 18.81 pre-B-cell line and hybrids derived by fusing 18.81 to the NSO myeloma fusion partner were transfected with a mu heavy-chain construct containing a nonsense mutation in the V region (Vn) or the constant region (Cn). Mutation was quantitated by reversion analysis using the ELISA spot assay to detect single cells secreting IgM. Fluctuation analysis revealed that V-region mutations spontaneously occurred in 18.81 cells at an average rate of 5.8 x 10(-6) per bp per cell generation and in selected 18.81-NSO hybrids at greatly increased rates of 1.6 x 10(-3) to 5.8 x 10(-4) per bp per generation. The Vn construct also reverted frequently in transgenic mice, indicating that it contained sufficient information to mutate at high rates both in vivo and in vitro. Sequence analysis of reverted genes revealed that reversion was due to point mutations. Since the rates and nature of the mutations that are occurring in these transfected genes are similar to those reported in vivo, it should be possible to use this system to identify the cis-acting sequences and trans-acting factors that are responsible for V-region somatic hypermutation.

Social status regulates growth rate: Consequences for life-history strategies

The life-history strategies of organisms are sculpted over evolutionary time by the relative prospects of present and future reproductive success. As a consequence, animals of many species show flexible behavioral responses to environmental and social change. Here we show that disruption of the habitat of a colony of African cichlid fish, Haplochromis burtoni (Günther) caused males to switch social status more frequently than animals kept in a stable environment. H. burtoni males can be either reproductively active, guarding a territory, or reproductively inactive (nonterritorial). Although on average 25–50% of the males are territorial in both the stable and unstable environments, during the 20-week study, nearly two-thirds of the animals became territorial for at least 1 week. Moreover, many fish changed social status several times. Surprisingly, the induced changes in social status caused changes in somatic growth. Nonterritorial males and animals ascending in social rank showed an increased growth rate whereas territorial males and animals descending in social rank slowed their growth rate or even shrank. Similar behavioral and physiological changes are caused by social change in animals kept in stable environmental conditions, although at a lower rate. This suggests that differential growth, in interaction with environmental conditions, is a central mechanism underlying the changes in social status. Such reversible phenotypic plasticity in a crucial life-history trait may have evolved to enable animals to shift resources from reproduction to growth or vice versa, depending on present and future reproductive prospects.

Circadian gating of cell division in cyanobacteria growing with average doubling times of less than 24 hours.

To ascertain whether the circadian oscillator in the prokaryotic cyanobacterium Synechococcus PCC 7942 regulates the timing of cell division in rapidly growing cultures, we measured the rate of cell division, DNA content, cell size, and gene expression (monitored by luminescence of the PpsbAI::luxAB reporter) in cultures that were continuously diluted to maintain an approximately equal cell density. We found that populations dividing at rates as rapid as once per 10 h manifest circadian gating of cell division, since phases in which cell division slows or stops recur with a circadian periodicity. The data clearly show that Synechococcus cells growing with doubling times that are considerably faster than once per 24 h nonetheless express robust circadian rhythms of cell division and gene expression. Apparently Synechococcus cells are able to simultaneously sustain two timing circuits that express significantly different periods.