The roles of antibody variable region hypermutation and selection in the development of the memory B-cell compartment.

Somatic hypermutation and selection of immunoglobulin (Ig) variable (V)-region genes, working in concert, appear to be essential for memory B-cell development in mammals. There has been substantial progress on the nature of the cis-acting DNA elements that regulate hypermutation. The data obtained suggest that the mechanisms of Ig gene hypermutation and transcription are intimately intertwined. While it has long been appreciated that stringent phenotypic selection forces are imposed on the somatically mutated Ig V regions generated during a T-cell dependent B-cell response, the mechanisms involved in this selection have remained enigmatic. Our studies have questioned the role of foreign antigen deposited on follicular dendritic cells in affinity-based positive selection of V regions, and have shown that this selection takes place in a "clone-autonomous" fashion. In addition, our data strongly suggest that affinity for antigen alone is not the driving force for selection of B-cell clones into the memory compartment. In contrast, we suggest that a combination of positive selection for increased foreign antigen binding, and negative selection of antibody V regions that are autoreactive at the onset of the response, or have acquired autoreactivity via hypermutation, results in the "specificity maturation" of the memory B-cell response.

Evaluation of loss and change of specificity resulting from random mutagenesis of an antibody VH region.

Most data available from in vivo sources regarding the impact of somatic hypermutation on Ab V region structure and function are heavily biased due to the influence of clonal selection. In an effort to address this issue directly, we "randomly" introduced point mutations throughout the length of the VH region of an anti-p-azophenylarsonate (Ars) Ab expressed as an Fab in the phage display format. This was accomplished by means of an error-prone PCR with two protocols, which resulted in two mutant libraries. The nature of the nucleotide substitutions obtained from each protocol differed from each other and resulted in different frequencies of phage clones that did not appear to contain Fab on their surfaces. However, the majority of mutants in both libraries lacked detectable Fab expression. Screening of the library containing the most expressed Fabs for those that had gained affinity for structurally related haptens yielded two independent mutants that lacked detectable affinity for Ars and had high affinity for p-azophenylsulfonate. These mutants both contained amino acid substitutions from Asn to Ser or Thr at VH CDR1 position 35, a putative Ars contact residue. In this paper, we discuss the significance of these data with regard to the frequencies of V region loss of function, gain of increased affinity, and gain of altered specificity that result from somatic hypermutation in vivo.

Random mutagenesis of two complementarity determining region amino acids yields an unexpectedly high frequency of antibodies with increased affinity for both cognate antigen and autoantigen.

To gain insight into the mechanism and limitations of antibody affinity maturation leading to memory B cell formation, we generated a phage display library of random mutants at heavy chain variable (V) complementarity determining region 2 positions 58 and 59 of an anti-p-azophenylarsonate (Ars) Fab. Single amino acid substitutions at these positions resulting from somatic hypermutation are recurrent products of affinity maturation in vivo. Most of the ex vivo mutants retained specificity for Ars. Among the many mutants displaying high Ars-binding activity, only one contained a position 58 and 59 amino acid combination that has been previously observed among the monoclonal antibodies (mAbs) derived from Ars-immunized mice. Affinity measurements on 14 of the ex vivo mutants with high Ars-binding activity showed that 11 had higher intrinsic affinities for Ars that the wild-type V region. However, nine of these Fabs also bound strongly to denatured DNA, a property neither displayed by the wild-type V region nor observed among the mutants characteristic of in vivo affinity maturation. These data suggest that ex vivo enhancement of mAb affinity via site-directed and random mutagenesis approaches may often lead to a reduction in antibody specificity that could complicate the use of the resulting mAbs for diagnostic and therapeutic applications. Moreover, the data are compatible with a hypothesis proposing that increased specificity for antigen, rather than affinity per se, is the driving force for formation of the memory B cell compartment.

In vitro synthesis of pp60v-src: myristylation in a cell-free system.

Covalent attachment of myristic acid to pp60v-src, the transforming protein of Rous sarcoma virus, was studied in a cell-free system. Using a synthetic peptide containing the first 11 amino acids of the mature pp60v-src polypeptide sequence as a substrate, we probed lysates from a variety of cells and tissues for N-myristyl transferase (NMT) activity. Nearly every eucaryotic cell type tested contained NMT, including avian, mammalian, insect, and plant cells. Since NMT activity was detected in rabbit reticulocyte lysates, we took advantage of the translational capability of these lysates to determine the precise point during translation at which myristate is attached to pp60v-src. src mRNA, transcribed from cloned v-src DNA, was translated in reticulocyte lysates which had been depleted of endogenous myristate. Addition of [3H]myristate to lysates 10 min after the start of synchronized translation resulted in a dramatic decrease in the incorporation of radiolabeled myristate into pp60v-src polypeptide chains. These results imply that although myristate can be attached posttranslationally to synthetic peptide substrates, myristylation in vivo is apparently a very early cotranslational event which occurs before the first 100 amino acids of the nascent polypeptide chain are polymerized.

xol-1: a gene that controls the male modes of both sex determination and X chromosome dosage compensation in C. elegans.

Loss-of-function mutations in the X-linked gene xol-1 cause the feminization and death of XO animals (normally males) by shifting the sex determination and dosage compensation pathways toward their hermaphrodite modes. XO-specific lethality most likely results from the reduction in X chromosome expression caused by xol-1 mutations. Mutations in genes required for the hermaphrodite mode of dosage compensation suppress lethality but not feminization, and restore X chromosome expression to nearly wild-type levels. Mutations in genes that control the hermaphrodite modes of both sex determination and dosage compensation fully suppress both defects. These interactions suggest that xol-1 is the earliest-acting gene in the known hierarchy controlling the male/hermaphrodite decision and is perhaps the gene nearest the primary sex-determining signal. We propose that the wild-type xol-1 gene product promotes male development by ensuring that genes (or gene products) directing hermaphrodite sex determination and dosage compensation are inactive in XO animals. Interestingly, in addition to feminizing XO animals, xol-1 mutations further masculinize XX animals already partially masculinized.

Assessment of X chromosome dosage compensation in Caenorhabditis elegans by phenotypic analysis of lin-14.

Caenorhabditis elegans compensates for the difference in X chromosome gene dose between males (XO) and hermaphrodites (XX) through a mechanism that equalizes the levels of X-specific mRNA transcripts between the two sexes. We have devised a sensitive and quantitative genetic assay to measure perturbations in X chromosome gene expression caused by mutations that affect this process of dosage compensation. The assay is based on quantitating the precocious alae phenotype caused by a mutation that reduces but does not eliminate the function of the X-linked gene lin-14. We demonstrate that in diploid animals the lin-14 gene is dosage compensated, implying that the normal dosage compensation mechanism in C. elegans lacks the capacity to compensate completely for the additional X chromosome in triplo-X animals. Using the lin-14 assay we compare the effects of mutations in the genes dpy-21, dpy-26, dpy-27, dpy-28, and dpy-22 on X-linked gene expression. Additionally, in the case of dpy-21 we correlate the change in phenotypic expression of lin-14 with a corresponding change in the lin-14 mRNA transcript level.

Caenorhabditis elegans compensates for the difference in X chromosome dosage between the sexes by regulating transcript levels.

The primary sex-determining signal in the nematode C. elegans is the ratio of X chromosomes to sets of autosomes (X/A ratio). As a consequence, males (XO; ratio 0.5) and hermaphrodites (XX; ratio 1.0) possess different doses of X-linked genes. Here we demonstrate that C. elegans compensates for this disparity in gene dose by equalizing the levels of X-specific mRNA transcripts in the two sexes. Moreover, we show that mutations in three autosomal genes disrupt the process of dosage compensation. Reduction in the activity of either dpy-21, dpy-27, or dpy-28 results in the overexpression of X-specific genes, 2- to 3-fold above wild-type levels.

Heat shock regulatory gene htpR influences rates of protein degradation and expression of the lon gene in Escherichia coli.

Upon a shift to high temperature, Escherichia coli increase their rate of protein degradation and also the expression of a set of "heat shock" genes. Nonsense mutants of htpR (also called hin), suppressed by a temperature-sensitive suppressor, show lower expression of heat shock genes at 30 degrees C and fail to respond to a shift to 42 degrees C. These mutants were found to have a lower capacity to degrade abnormal or incomplete proteins than that of wild-type cells. This reduction in proteolysis equals or exceeds that in lon mutants, which encode a defective ATP-dependent protease, protease La, and is particularly large in htpR lon double mutants. The activity of protease La was higher in wild-type cells than in htpR mutants grown at 30 degrees C and increased upon shift to 42 degrees C only in the wild type. To determine whether htpR influences transcription of the lon gene, a lon-lacZ operon fusion was utilized. Introduction of the htpR mutation reduced transcription from the lon promoter at 30 degrees C and 37 degrees C. This defect was corrected by a plasmid (pFN97) carrying the wild-type htpR allele. Induction of the heat shock response with ethanol had little or no effect in htpR mutants but stimulated lon transcription 2-3 fold in wild-type cells and htpR cells carrying pFN97. Thus, lon appears to be a heat shock gene, and increased synthesis of protease La under stressful conditions may help to prevent the accumulation of damaged cellular protein.

Anaerobiosis induces expression of ant, a new Escherichia coli locus with a role in anaerobic electron transport.

Escherichia coli has a formate hydrogenlyase system which allows it to maintain an electron balance during anaerobic growth by passing electrons from formate to H+ ions, thus generating H2. The Mu d1(Ap lac) bacteriophage was used to generate mutants that were defective in passing electrons from formate to benzyl viologen, an artificial electron acceptor. A subset of these mutants was studied in which beta-galactosidase was expressed at much higher levels under anaerobic conditions than under aerobic conditions. If nitrate was present during anaerobic growth, the same levels of beta-galactosidase were seen in these fusion strains as were seen under aerobic conditions. The Mu d1(Ap lac) insertions in these mutants were genetically mapped between mutS and srl and thus define a new locus we have termed ant (anaerobic electron transport). Recombinant lambda derivatives were isolated which complemented the deficiency of the ant mutants in anaerobic electron transport and also carried a trans-acting region of DNA which reduced expression of the ant-lac fusions under anaerobic conditions; a probe to the ant region was generated from one of these recombinant lambda derivatives. Southern hybridization analysis revealed that the four independent ant::Mu d1(Ap lac) fusions we isolated spanned an approximately 5-kilobase region and that all were transcribed in the same direction, counterclockwise on the E. coli genetic map.