Mouse immunoglobulin gene rearrangements. The sequence of a nonexpressed lambda 3-chain gene

A functionally defective lambda 3-immunoglobulin chain gene has been cloned from plasmacytoma HOPC-1 (gamma 2b, lambda 1). The lambda 3 gene resulted from the juxtaposition of the germline V lambda 1 sequence with a J lambda 3 C lambda 3 gene segment. DNA sequencing of the rearranged V lambda 1 J lambda 3 exon showed the presence of a single base pair deletion at the site of V-J joining. The alteration in the reading frame caused by this deletion generated a stop codon at the 3' end of J lambda 3, thus rendering this gene nonfunctional for light chain production. In addition, a one-point mutation in the J lambda 3-C lambda 3 intron distinguishes the rearranged gene from the unrearranged counterpart. The implications that this rearrangement has in terms of the mechanism of somatic mutations and of selective proliferation of B cells mediated by antigen stimulation are discussed.     

A stochastic model for chemotaxis based on the ordered extension of pseudopods

Many amoeboid cells move by extending pseudopods. Here I present a new stochastic model for chemotaxis that is based on pseudopod extensions by Dictyostelium cells. In the absence of external cues, pseudopod extension is highly ordered with two types of pseudopods: de novo formation of a pseudopod at the cell body in random directions, and alternating right/left splitting of an existing pseudopod that leads to a persistent zig-zag trajectory. We measured the directional probabilities of the extension of splitting and de novo pseudopods in chemoattractant gradients with different steepness. Very shallow cAMP gradients can bias the direction of splitting pseudopods, but the bias is not perfect. Orientation of de novo pseudopods require much steeper cAMP gradients and can be more precise. These measured probabilities of pseudopod directions were used to obtain an analytical model for chemotaxis of cell populations. Measured chemotaxis of wild-type cells and mutants with specific defects in these stochastic pseudopod properties are similar to predictions of the model. These results show that combining splitting and de novo pseudopods is a very effective way for cells to obtain very high sensitivity to stable gradient and still be responsive to changes in the direction of the gradient.     

Photoreceptor disc morphogenesis: The classical evagination model prevails

Vision begins in photoreceptor outer segments with light captured by opsins in continually synthesized disc membranes. The process by which rod photoreceptor discs are formed has been controversial. In this issue, Ding et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201508093) show conclusively that rod discs are formed by plasma membrane evagination.The vertebrate retina contains two types of photoreceptors, rod cells and cone cells, whose outer segments initiate phototransduction under night and daytime conditions, respectively. The outer segments of these cells lack ER, Golgi, and mitochondria and are filled with hundreds to a few thousand flattened membrane organelles, called photoreceptor discs, which are loaded with the molecular machinery of phototransduction. The structural organization of outer segments differs between rods and cones. Although cone outer segments contain “open” discs that are infoldings of the plasma membrane, rod outer segments possess “closed” discs that are completely separated from the plasma membrane.In 1967, in a paper that has been cited nearly 800 times, Richard Young reported the seminal finding that rod and cone outer segments are continually renewed (Young, 1967). Young’s classic experiment was elegantly simple: he injected [³H]methionine into a rat, mouse, and frog and performed autoradiograms of the excised retina on various days after the injection. He observed that the radiolabeled band moved along the outer segment as time after injection increased and ultimately disappeared at the apex of the cell (Fig. 1, republished from Young, 1967). (As Young was at the University of California, Los Angeles, this result was given the memorable moniker of “the UCLA marching band.”) Young’s seminal insight that outer segments are continually rebuilt posed a problem that has challenged photoreceptor cell biologists ever since: How are rod disc membranes initially formed? In this issue, Ding et al. present a compelling resolution to this question. Specifically, their work differentiates between currently competing models to determine whether rod discs are formed by evagination of plasma membrane at the base of the outer segment or by fusion of intracellular vesicles transported to the outer segment.Open in a separate windowFigure 1.Photoreceptor outer segments are continually renewed. Rats were injected with [³H]methionine, and radioautographs of photoreceptor cells were performed on various days after the injection. As time after injection increases (images 2–7), the radiolabel components are displaced from the inner segment along the outer segment toward the apex of the cell, revealing that the outer segment is continually renewed (figure republished from Young, 1967).The classic hypothesis of disc morphogenesis is that they are formed by evagination of basal outer segment plasma membrane (Steinberg et al., 1980). This hypothesis is based largely on evidence that one surface of the most basal discs of rods is open to the extracellular space, as shown by EM (Carter-Dawson and LaVail, 1979; Steinberg et al., 1980), with lipophilic dye fluorescence (Laties et al., 1976), and by analysis of membrane capacitance (Rüppel and Hagins, 1973). In addition, rods and cones might be expected to share a common machinery of disc formation. Because most cone discs are well established by EM, lipophilic dye imaging, and electrophysiology to be continuous with the plasma membrane, nascent rod discs would seem likely to also be part of the plasma membrane. Thus, according to the classic hypothesis, new discs in both photoreceptor types are formed from outgrowths (evaginations) of the plasma membrane at the outer segment base. In both photoreceptor types, discs would begin life with one face exposed to the extracellular space, but at some point after formation, rod discs would pinch off from the outer segment plasma membrane to become self-contained and fully separated from the plasma membrane, whereas cones discs remain open. On the contrary, the vesicle fusion hypothesis postulates that nascent discs are born completely internalized in rods. Photoreceptor outer segments are now understood to be the plus end of a modified primary cilium (Bloodgood, 2009) and are joined to their inner segments by a narrow ciliary tube called the connecting cilium. This realization, combined with evidence of vesicles in the connecting cilium seen in electron micrographs, has been taken to support the model that vesicles are actively transported through the connecting cilium and generate nascent discs by membrane fusion at the base of the outer segment (Chuang et al., 2007, 2015).Ding et al. (2015) addressed these competing hypotheses with two distinct approaches. First, they treated sections of retinas of mice perfused with a membrane-staining mixture of tannic acid and uranyl acetate and performed EM. Because tannic acid penetrates intact membranes poorly, this treatment distinguishes between membranes exposed to the extracellular space and intracellular membrane structures. The researchers found that, like the plasma membrane, a small number of basal rod discs were intensely stained by tannic acid, whereas the staining of fully internalized discs was weak, confirming that newly formed rod discs are open to the extracellular space. Consistently and strikingly, EM analysis also revealed a single basal disc face (approximately five to seven discs north of the most basal disc) that is contiguous with the plasma membrane. Second, Ding et al. (2015) performed EM with an immunogold-tagged antibody raised against an intracellular epitope of peripherin, a protein that plays an essential role in disc stacking (Arikawa et al., 1992; Goldberg, 2006). Quantification of gold particle counts showed that the peripherin antibody closely associated intracellularly with the edges of fully internalized discs but was negligibly associated with the surface of nascent discs identified as facing the extracellular space, suggesting that peripherin redistributes along the rod disc edge upon its separation from the plasma membrane and enclosure into the outer segment. Finally, Ding et al. (2015) performed experiments using the fixation techniques reported by other investigators and demonstrated that artifacts of tissue fixation were responsible for the erroneous interpretation that basal discs are fully internalized and for the evidence supporting the vesicular fusion hypothesis.Other tools, such as superresolution microscopy of living rods stained with lipophilic dyes or fluorescent antibodies raised against epitopes on the extracellular face of the rod plasma membrane, could further test aspects of the evagination model of disc formation. Nonetheless, the work of Ding et al. (2015) unequivocally shows that basal rod discs are open to the extracellular space and provides a new system and conceptual framework for the investigation of the fundamental biological mechanism of plasma membrane evagination. As outer segment discs exhibit a specialized composition of lipids and phototransduction proteins, further work will also focus on how disc lipids and proteins are transported from the inner segment to the basal outer segment. The current hypotheses about such transport include (a) vesicular transport through the connecting cilium followed by fusion with the outer segment plasma membrane; (b) directed transport through the connecting cilium membrane after vesicle fusion at the base of the connecting cilium in the inner segment; and (c) exocytotic release from the inner segment followed by endocytotic capture in the outer segment. As the molecular details of disc formation and specialization become clearer, Richard Young’s “UCLA marching band” (Young, 1967) will continue to have a broad conceptual impact on the cell biology of photoreceptor development and cilia.     

A simple stochastic gene substitution model

If the fitnesses of n haploid alleles in a finite population are assigned at random and if the alleles can mutate to one another, and if the population is initially fixed for the kth most fit allele, then the mean number of substitutions that will occur before the most fit allele is fixed is shown to be

$\text{1}\text{2}\text{+}\text{1}\text{k}\text{+}\text{∑}\text{i=2}\text{k?1}\text{(i+3)}\text{(2i(i+1))}$

when selection is strong and mutation is weak. This result is independent of the parameters that went into the model. The result is used to provide a partial explanation for the large variance observed in the rates of molecular evolution.     

Gene targeting in the Ig kappa locus: efficient generation of lambda chain-expressing B cells, independent of gene rearrangements in Ig kappa.

The production of lambda chain-expressing B cells was studied in mice in which either the gene encoding the constant region of the kappa chain (C kappa) or the intron enhancer in the Ig kappa locus was inactivated by insertion of a neomycin resistance gene. The two mutants have similar phenotypes: in heterozygous mutant mice the fraction of lambda chain-bearing B cells is twice that in the wildtype. Homozygous mutants produce approximately 7 times more lambda-expressing B cells (and about 2.3 times fewer total B cells) in the bone marrow than their normal counterparts, suggesting that B cell progenitors can differentiate into either kappa- or lambda-producing cells and do the latter in the mutants. Whereas gene rearrangements in the Ig kappa locus are blocked in the case of enhancer inactivation, they still occur in that of the C kappa mutant, although in this mutant RS rearrangement is lower than in the wildtype. This indicates that gene rearrangements in the Ig lambda locus can occur in the absence of a putative positive signal resulting from gene rearrangements in Ig kappa, including RS recombination. Complementing these results, we also present data indicating that in normal B cell development kappa chain rearrangement can be preceded by lambda chain rearrangement and that the frequency of kappa/lambda double producers is small and insufficient to explain the massive production of lambda chain-expressing B cells in the mutants.     

The OR control system of bacteriophage lambda. A physical-chemical model for gene regulation

A quantitative model has been developed for processes in the bacteriophage lambda that control the switchover from lysogenic to lytic modes of growth. These processes include the interactions of cI repressor and cro proteins at the three DNA sites of the right operator, OR, the binding of RNA polymerase at promoters PR and PRM, the synthesis of cI repressor and cro proteins, and the degradative action of recA during induction of lysis. The model is comprised of two major physical-chemical components: a statistical thermodynamic theory for relative probabilities of the various molecular configurations of the control system; and a kinetic model for the coupling of these probabilities to functional events, including synthesis of regulatory proteins cI and cro. Using independently evaluated interaction constants and rate parameters, the model was found capable of predicting essential physiological characteristics of the system over an extended time. Sufficiency of the model to predict known physiological properties lends credence to the physical-chemical assumptions used in its construction. Several major physiological characteristics were found to arise as "system properties" through the non-linear, time-dependent, feedback-modulated combinations of molecular interactions prescribed by the model. These include: maintenance of the lysogenic state in the absence of recA-mediated cI repressor degradation; induction of lysis and the phenomenon of subinduction; and autogenous negative control of cro. We have used the model to determine the roles, within the composite system, of several key molecular processes previously characterized by studies in vitro. These include: co-operativity in cI repressor binding to DNA; interactions between repressors and RNA polymerase (positive control); and the monomer-dimer association of cI repressor molecules. A major role of cI repressor co-operativity is found to be that of guaranteeing stability of the lysogenic state against minor changes in cI repressor levels within the cell. The role of positive control seems to be that of providing for a peaked, rather than monotonic, dependence of PRM activity on cI repressor level, while permitting PR activity to be a step function. The model correlates an immense body of studies in vivo and in vitro, and it makes testable predictions about molecular phenomena as well as physiological characteristics of bacteriophage lambda. The approach developed in this study can be extended to include more features of the lambda system and to treat other systems of gene regulation.     

A kinetic model for bacterial transfection: the lambda DNA system.

A kinetic model describing the binding and uptake of free lambda phage DNA by the bacterium Escherichia coli is presented. The model is based on the assumption that adsorbed ‘helper’ phage particles serve as functional sites to which the lambda DNA specifically binds. When applied to experimental data, the model describes the reaction between cells and DNA as a rapid binding of DNA to helper phage attachment sites, followed by a slow, irreversible incorporation of bound DNA into the cells. Features of the model include a time-dependent exponential decay of functional sites required for DNA uptake and a minimum time for irreversibly bound DNA to enter the cell. We suggest that this model may be useful in studying processes involved in the active transport of DNA across a permeability barrier.     

Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor

Wiesmeyer, Herbert (Vanderbilt University, Nashville, Tenn.). Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor. J. Bacteriol. 91:89-94. 1966.-The concentration of lambda repressor molecules within a lambda lysogenic cell was estimated from the multiplicity of superinfecting homologous phage necessary to permit replication and release of plaque-forming units. A multiplicity of 20 superinfecting phage was found sufficient to permit replication to occur in the normal lambda lysogen. The phage released after lysis of the superinfected lysogen was composed of both prophage and superinfecting phage types. Superinfection of the lysogen at lower multiplicities resulted in the lysis of only a small percentage of infected cells and is thought to represent a possible heterogeneity of repressor concentration in the lysogenic population. Viability of the superinfecting particle was found to be unnecessary for titration of the repressor. The repressor concentration in three lysogens of the nonultraviolet-inducible mutant of lambda, lambda(ind-), was found to be greater than 20 regardless of the host bacterium. However, the number of cells yielding phage after superinfection was found to vary with the particular host. The specificity of the lambda repressor was shown to be limited to homologous phage, as determined following heterologous superinfection experiments with phages T6r, 82c, 434c, 434hy, and 424. In all instances except that of superinfection with phage 434hy, only heterologous phage replication occurred. Superinfection by phage 434hy resulted in the release of both prophage and superinfecting phage types. The latter type represented approximately 80% of the total phage released.     

Conservation of the immunoglobulin C lambda 5 gene in the Mus gene.

A gene encoding the lambda 5 light chain constant region was isolated from a genomic library from the SPE mouse strain (C lambda 5S). SPE is an inbred wild mouse strain belonging to the Mus 3 or Mus spretus group that has been genetically isolated from Mus 1 (the group to which laboratory mice belong) for a period of 1-3 million years. The sequence of the C lambda 5S gene shows strong homology to C lambda 5 of (C57BL/6J x DBA/2)F1 both in the coding region (98% identity) and in the 5'- and 3'-flanking regions (98 and 95% identity, respectively). Sequence comparison of C lambda 5 genes with C lambda 1 of BALB/c shows only few substitutions in the C lambda 5 coding regions and suggests that the three genes have a common ancestor. These data indicate that the C lambda 5 gene has evolved under strong selective pressure and probably encodes a functional gene product. The conservation of the C lambda 5 gene in various Mus species was observed by high stringency Southern blot analyses using a C lambda 5S probe on DNA sample from members of four different groups of wild mice. All the laboratory and wild mouse strains tested, including those with amplified sets of C lambda 1 and C lambda 2 hybridizing sequences, showed only single C lambda 5 hybridizing fragments. Little variation in size of restriction fragments detected with the C lambda 5 probe was seen in the different Mus species suggesting a high degree of conservation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lethal action of bacteriophage lambda S gene.

The functions of the bacteriophage lambda lysis genes S, R, and Rz were investigated. Different combinations of wild-type and inactive alleles of all three lysis genes were cloned into the plasmid pBH20 and were expressed under the control of a lac operator-promoter. The involvement of the Rz gene in lysis was proposed in our previous work and was confirmed by the Mg2+-dependent lysis defect of clones in which part of the Rz gene is deleted. Membrane vesicles prepared from induced S+ cells were shown to have a severely reduced capacity for active transport of glucose; this defect was detectable at least 20 min before lysis. Cell viability was also shown to decrease very soon after induction, long before physiological death and lysis; this decrease in viability is absolutely dependent on S expression and independent of R and Rz. The nonviable fraction of cells at any time after induction was demonstrated to be equal to the fraction committed to eventual lysis. Induction of an Sts clone showed that the S gene product is stable and capable of inducing lysis long after the cessation of synthesis of S gene product. A model for S action is proposed.     

Condensation prevails over B-A transition in the structure of DNA at low humidity

B-A transition and DNA condensation are processes regulated by base sequence and water activity. The constraints imposed by interhelical interactions in condensation compromise the observation of the mechanism by which B and A base-stacking modes influence the global state of the molecule. We used a single-molecule approach to prevent aggregation and mechanical force to control the intramolecular chain association involved in condensation. Force-extension experiments with optical tweezers revealed that DNA stretches as B-DNA under ethanol and spermine concentrations that favor the A-form. Moreover, we found no contour-length change compatible with a cooperative transition between the A and B forms within the intrinsic-force regime. Experiments performed at constant force in the entropic-force regime with magnetic tweezers similarly did not show a bistable contraction of the molecules that could be attributed to the B-A transition when the physiological buffer was replaced by a water-ethanol mixture. A total, stepwise collapse was found instead, which is characteristic of DNA condensation. Therefore, a low-humidity-induced change from the B- to the A-form base-stacking alone does not lead to a contour-length shortening. These results support a mechanism for the B-A transition in which low-humidity conditions locally change the base-stacking arrangement and globally induce DNA condensation, an effect that may eventually stabilize a molecular contour-length reduction.     

Quality prevails over identity in the sexually selected vocalisations of an ageing mammal

Background

Male sexually selected vocalisations generally contain both individuality and quality cues that are crucial in intra- as well as inter-sexual communication. As individuality is a fixed feature whereas male phenotypic quality changes with age, individuality and quality cues may be subjected to different selection pressures over time. Individuality (for example, morphology of the vocal apparatus) and quality (for example, body size and dominance status) can both affect the vocal production mechanism, inducing the same components of vocalisations to convey both kinds of information. In this case, do quality-related changes to the acoustic structure of calls induce a modification of vocal cues to identity from year to year? We investigated this question in fallow deer (Dama dama), in which some acoustic parameters of vocalisations (groans) code for both individuality and quality.

Results

We carried out a longitudinal analysis of groan individuality, examining the effects of age and dominance rank on the acoustic structure of groans of the same males recorded during consecutive years. We found both age- and rank-related changes to groans; the minimum values of the highest formant frequencies and the fundamental frequency increased with the age of males and they decreased when males became more dominant. Both age- and rank-related acoustic parameters contributed to individuality. Male quality changed with age, inducing a change in quality-related parameters and thus, a modification of vocal cues to male individuality between years.

Conclusions

The encoding of individuality and quality information in the same components of vocalisations induces a tradeoff between these two kinds of signals over time. Fallow deer vocalisations are honest signals of quality that are not fixed over time but are modified dynamically according to male quality. As they are more reliable cues to quality than to individuality, they may not be used by conspecifics to recognize a given male from one year to another, but potentially used by both sexes to assess male quality during each breeding season.

     

Engineered internal noise stochastic resonator in gene network: a model study

Based on a genetic bistable switch model coupled with a gene oscillator model, we have constructed a mesoscopic stochastic model for the coupled synthetic gene network, and studied how internal noise would influence the oscillation of such a system. We found that the state-to-state transitions can occur if the internal noise is taken into account, and the performance of resulting oscillation can reach a maximum in a certain internal noise level, which indicates the occurrence of internal noise stochastic resonance (SR) and makes the coupled gene network work as a stochastic resonator. The potential role of such an effect on gene expression systems is also discussed.     

A coliphage lambda vector with enhanced biological containment: lambda gtALO.lambda B.

The biological containment of the lambda gt family of cloning vectors has been enhanced by conditionally blocking DNA replication as well as head and tail morphogenesis. The vector, lambda gtALO.lambda B, was constructed by crossing the Oam29, Aama1 and Lam439 mutations into lambda gt.lambda B. The mutation blocking phage DNA replication, Oam29, is suppressed by suII+ or suIII+. The head gene mutation, Aama1, is suppressed by suIII+ but not by suII+ and the tail gene mutation, Lam439, is suppressed by suII+ but not by suIII+. This allows the option of increasing the biological containment by producing heads when a large amount of cloned DNA is being prepared from an individual isolate. A model recombinant, lambda gt Aama1 Lam439 Oam29.KmR' (lambda gtALO.KmR') was constructed and the containment of the vector was evaluated by the series of standardized experiments required for EK2 certification.