Analysis of microsatellite instability and hypermutation of immunoglobulin variable genes in Werner syndrome.

Werner syndrome (WS) is a human premature aging syndrome, which is associated with high frequencies of neoplasia and genetic instability. We have examined the occurrence of microsatellite instability, which may result from defective mismatch repair, in lymphoblastoid cell lines derived from nine WS patients. Instability was measured at the D2S123 locus by gel analysis of PCR products. Three WS cell lines had 4-13% altered alleles, compared with 0% in the other six lines. The increased frequency of microsatellite instability could not readily be associated with overt cancer or any other known clinical condition in the three patients. To examine whether the WS defect affected the humoral immune system, we measured the hypermutation of immunoglobulin variable genes in peripheral blood cells from the WS patient who donated the cell line with the highest frequency of microsatellite instability. The frequency and pattern of mutation was similar to that from normal individuals, suggesting that the Werner protein is not involved in generating hypermutation.

Third complementarity-determining region of mutated VH immunoglobulin genes contains shorter V, D, J, P, and N components than non-mutated genes.

The third complementarity-determining region (CDR3) of immunoglobulin variable genes for the heavy chain (VH) has been shown to be shorter in length in hypermutated antibodies than in non-hypermutated antibodies. To determine which components of CDR3 contribute to the shorter length, and if there is an effect of age on the length, we analysed 235 cDNA clones from human peripheral blood of VH6 genes rearranged to immunoglobulin M (IgM) constant genes. There was similar use of diversity (D) and joining (JH) gene segments between clones from young and old donors, and there was similar use of D segments among the mutated and non-mutated heavy chains. However, in the mutated heavy chains, there was increased use of shorter JH4 segments and decreased use of longer JH6 segments compared to the non-mutated proteins. The overall length of CDR3 did not change with age within the mutated and non-mutated categories, but was significantly shorter by three amino acids in the mutated clones compared to the non-mutated clones. Analyses of the individual components that comprise CDR3 indicated that they were all shorter in the mutated clones. Thus, there were more nucleotides deleted from the ends of VH, D, and JH gene segments, and fewer P and N nucleotides added. The results suggest that B cells bearing immunoglobulin receptors with shorter CDR3s have been selected for binding to antigen. A smaller CDR3 may allow room in the antibody binding pocket for antigen to interact with CDRs 1 and 2 as well, so that as the VDJ gene undergoes hypermutation, substitutions in all three CDRs can further contribute to the binding energy.

Impact of age on hypermutation of immunoglobulin variable genes in humans.

Chronological aging is associated with an accumulation of DNA mutations that results in cancer formation. The effect of aging on spontaneous mutations in humans is difficult to study because mutations are infrequent in the overall genome and tumors are relatively rare. In contrast, somatic mutations in immunoglobulin variable genes are abundant and can be studied in peripheral blood lymphocytes. To determine if aging alters the frequency and pattern of hypermutation, we sequenced 331 cDNA clones with rearranged V(H)6 genes and compared 452 mutations from young humans to 570 mutations from old humans. There were more mutated clones in the young population compared to the old population. Among the mutated clones, the frequency, location, and types of substitutions were similar between the young and the old groups. However, the ratio of replacement-to-silent mutations was much higher in the complementarity-determining regions of heavy chains from old people, which indicates that their B cells had been selected by antigen. Among individuals, there was variability in the frequency of tandem mutations, which we have observed in mice defective for the PMS2 mismatch repair protein. Microsatellite variability in DNA, which is caused by impaired mismatch repair, was then measured, and there was a strong correlation between the frequency of tandem mutations and microsatellite alterations. The data suggest that individuals vary in their mismatch repair capacity, which can affect the mutational spectra in their antibodies.

DNA polymerase eta is an A-T mutator in somatic hypermutation of immunoglobulin variable genes.

To determine whether DNA polymerase eta plays a role in the hypermutation of immunoglobulin variable genes, we examined the frequency and pattern of substitutions in variable VH6 genes from the peripheral blood lymphocytes of three patients with xeroderma pigmentosum variant disease, whose polymerase eta had genetic defects. The frequency of mutation was normal but the types of base changes were different: there was a decrease in mutations at A and T and a concomitant rise in mutations at G and C. We propose that more than one polymerase contributes to hypermutation and that if one is absent, others compensate. The data indicate that polymerase eta is involved in generating errors that occur predominantly at A and T and that another polymerase(s) may preferentially generate errors opposite G and C.

Altered spectra of hypermutation in DNA repair-deficient mice.

Affinity maturation of the humoral immune response is based on the ability of immunoglobulin variable genes to undergo a process of rapid and extensive somatic mutation followed by antigenic selection for antibodies with higher affinity. While the behaviour of this somatic hypermutation phenomenon has been well characterized over the last 20 years, the molecular mechanism responsible for inserting mutations has remained shrouded. To better understand this mechanism, we studied the interplay between hypermutation and other DNA associated activities such as DNA repair. There was no effect on the frequency and pattern of hypermutation in mice deficient for nucleotide excision repair, base excision repair and ataxia-telangiectasia mutated gene repair of double strand breaks. However, variable genes from mice lacking some components of mismatch repair had an increased frequency of tandem mutations and had more mutations of G and C nucleotides. These results suggest that the DNA polymerase(s) involved in the hypermutation pathway produces a unique spectra of mutations, which is then altered by mismatch repair and antigenic selection. We, also describe the differential pattern of expression of some nuclear DNA polymerases in hypermutating versus non-hypermutating B lymphocytes. The rapidly dividing germinal centre B cells expressed DNA polymerases alpha, beta, delta, epsilon and zeta, whereas the resting non-germinal centre cells did not express polymerases alpha or epsilon at detectable levels, although they did express polymerases beta, delta and zeta. The lack of expression of polymerase epsilon in the non-germinal centre cells suggests that this enzyme has a critical role in chromosomal replication but does not participate in DNA repair in these cells.

Differential expression of DNA polymerase epsilon in resting and activated B lymphocytes is consistent with an in vivo role in replication and not repair.

DNA polymerases may be differentially expressed by cells during periods of quiescence and proliferation. Murine B cells are an ideal population to study because their division time varies widely in vivo, and different subsets can be easily isolated. Consequently, we analyzed RNA from resting cells (B220(+)peanut agglutinin(-)) and activated germinal center cells (B220(+)peanut agglutinin(+)) from spleens by reverse transcriptase-PCR using primers for five nuclear polymerases and their associated subunits. Gel analyses of the amplified products showed that the rapidly-dividing germinal center B cells expressed DNA polymerases alpha, beta, delta, epsilon, and zeta. The resting B cells did not express polymerases alpha or epsilon at detectable levels, although they did express polymerases beta, delta, and zeta. Thus, polymerase epsilon, as well as alpha, appears to have a primary role in chromosomal replication of murine B lymphocytes. Further, the lack of expression of polymerase epsilon in resting cells indicates that this enzyme is not used in any DNA repair pathways by these cells. The expression of polymerase zeta by resting cells suggests that it has another role in DNA repair, perhaps recombination, in addition to its function of bypassing damage during chromosomal replication.

Hypermutation in Ig V genes from mice deficient in the MLH1 mismatch repair protein.

During somatic hypermutation of Ig V genes, mismatched nucleotide substitutions become candidates for removal by the DNA mismatch repair pathway. Previous studies have shown that V genes from mice deficient for the MSH2 and PMS2 mismatch repair proteins have frequencies of mutation that are comparable with those from wild-type (wt) mice; however, the pattern of mutation is altered. Because the absence of MSH2 and PMS2 produced different mutational spectra, we examined the role of another protein involved in mismatch repair, MLH1, on the frequency and pattern of hypermutation. MLH1-deficient mice were immunized with oxazolone Ag, and splenic B cells were analyzed for mutations in their V kappa Ox1 light chain genes. Although the frequency of mutation in MLH1-deficient mice was twofold lower than in wt mice, the pattern of mutation in Mlh1-/- clones was similar to wt clones. These findings suggest that the MLH1 protein has no direct effect on the mutational spectrum.

Homogeneous rate of degradation of nuclear DNA during apoptosis.

DNA fragmentation during apoptosis is characterized by endonucleolytic cleavage of chromosomal DNA into an oligonucleosomal ladder. To determine if actively transcribed genes are more susceptible to cleavage during apoptosis than non-transcribed genes, the rate of fragmentation of differentially expressed genes was measured in B-lymphocyte hybridoma cells. Five genes were studied based on their transcriptional activity and/or nuclear localization, and mitochondrial DNA was assayed as a negative control for apoptotic fragmentation. Apoptosis was induced in the hybridoma cells by ultraviolet light, and DNA was prepared at multiple time points after ultraviolet irradiation. Degradation into an oligonucleosomal ladder appeared as early as 2 h after treatment, showing that fragmentation is rapidly activated in hybridoma cells. The DNA was then digested with restriction enzymes, separated by gel electrophoresis and hybridized with the gene-specific probes for Southern blot analyses. Loss of gene-specific signals was measured by quantitation of autoradiographs. The results show all of the nuclear genes were degraded at the same rate regardless of their transcriptional status or nuclear localization. The data suggest that once the cell activates its destruction program, nuclear DNA is rapidly degraded in a homogeneous manner.

Increased hypermutation at G and C nucleotides in immunoglobulin variable genes from mice deficient in the MSH2 mismatch repair protein.

Rearranged immunoglobulin variable genes are extensively mutated after stimulation of B lymphocytes by antigen. Mutations are likely generated by an error-prone DNA polymerase, and the mismatch repair pathway may process the mispairs. To examine the role of the MSH2 mismatch repair protein in hypermutation, Msh2-/- mice were immunized with oxazolone, and B cells were analyzed for mutation in their VkappaOx1 light chain genes. The frequency of mutation in the repair-deficient mice was similar to that in Msh2+/+ mice, showing that MSH2-dependent mismatch repair does not cause hypermutation. However, there was a striking bias for mutations to occur at germline G and C nucleotides. The results suggest that the hypermutation pathway frequently mutates G.C pairs, and a MSH2-dependent pathway preferentially corrects mismatches at G and C.

Altered spectra of hypermutation in antibodies from mice deficient for the DNA mismatch repair protein PMS2.

Mutations are introduced into rearranged Ig variable genes at a frequency of 10(-2) mutations per base pair by an unknown mechanism. Assuming that DNA repair pathways generate or remove mutations, the frequency and pattern of mutation will be different in variable genes from mice defective in repair. Therefore, hypermutation was studied in mice deficient for either the DNA nucleotide excision repair gene Xpa or the mismatch repair gene Pms2. High levels of mutation were found in variable genes from XPA-deficient and PMS2-deficient mice, indicating that neither nucleotide excision repair nor mismatch repair pathways generate hypermutation. However, variable genes from PMS2-deficient mice had significantly more adjacent base substitutions than genes from wild-type or XPA-deficient mice. By using a biochemical assay, we confirmed that tandem mispairs were repaired by wild-type cells but not by Pms2(-/-) human or murine cells. The data indicate that tandem substitutions are produced by the hypermutation mechanism and then processed by a PMS2-dependent pathway.

Dual enigma of somatic hypermutation of immunoglobulin variable genes: targeting and mechanism.

The immunoglobulin loci are uniquely unstable regions of the genome which undergo as much mutation and selection in a matter of days as a species can undergo in generations of evolution. We have studied the mutational pattern and targeting of this unusual hypermutation process over the past 16 years. The pattern of somatic mutations in rearranged variable (V) genes differs from the pattern of meiotic mutations, indicating that a different mechanism generates hypermutation than generates spontaneous mutation. Hypermutations begin on the 5' end of rearranged V genes downstream of the transcription initiation site and continue through the V exon and into the 3'-flanking region before tapering off. Mutations are located randomly throughout the DNA sequence and exhibit strand bias. The targeting of mutations to the region in and around the rearranged V gene appears to require interactions between the promoter and downstream intronic DNA sequences. The same mechanism that initiates hypermutation around V genes may also produce double-strand breaks that catalyze homologous recombination between rearranged V genes on two chromosomal alleles. With this data we have built a model of hypermutation which predicts that V-region DNA is destabilized at the nuclear matrix during transcription and undergoes strand breaks.

Insertion of 2 kb of bacteriophage DNA between an immunoglobulin promoter and leader exon stops somatic hypermutation in a kappa transgene.

Somatic hypermutation in rearranged immunoglobulin variable genes occurs in a 2kb region of DNA that is delimited on the 5' side by the promoter and on the 3' side by intron DNA. To identify sequence features that activate the mutation mechanism, we increased the distance between the promoter and the leader region to test whether the spacing of these elements was important. The promoter was separated from the leader sequence by inserting a 2 kb fragment of noncoding bacteriophage lambda DNA between the TATA box and ATG initiator codon in a kappa transgene. Mice from three founder lines were immunized, RNA and DNA were isolated from spleen and Peyer's patch B cells, and transcription of the transgene was confirmed. The frequency of mutation in endogenous heavy chain genes was high, indicating that some B cells underwent hypermutation. However, no hypermutation was found in the transgenic bacteriophage or variable region sequences. Hypermutation did occur in another kappa transgene that had a deletion of the VJ coding sequence, showing that the basic construct is functional and that the VJ exon is not necessary for the mutation mechanism. It is likely that the bacteriophage sequence is a potential substrate for mutation because other heterologous sequences have been shown to undergo mutation if placed downstream of the leader exon. The results suggest that the promoter should be contiguous with the leader exon for the mutation mechanism to function.

Conventional color Doppler velocity sonography versus color Doppler energy sonography for the diagnosis of acute experimental torsion of the spermatic cord.

OBJECTIVE: We compared color Doppler velocity sonography and color Doppler energy sonography for the diagnosis of spermatic cord torsion in a canine model and determined the degree of torsion necessary to acutely halt testicular blood flow. MATERIALS AND METHODS: Spermatic cord torsion was created in five dogs by exposing and rotating the ipsilateral testis 0 degree, 180 degrees, 270 degrees, 360 degrees, 450 degrees, and 540 degrees. Detorsion followed. The testicles were scanned at each torsion stop using both color Doppler velocity sonography and color Doppler energy sonography. Doppler parameters were optimized (by phantom and test scans) and maintained at a tolerable noise level throughout the experiment. Readers who were unaware of the degree of torsion compared flow in the rotated and contralateral control testes. RESULTS: Flow became undetectable by color Doppler velocity sonography and color Doppler energy sonography at 450 degrees in four of five cases and at 540 degrees in one of five cases. We found no significant difference between the velocity and the energy techniques for detecting this absence of flow (p > .05, Wilcoxon test). We found a significant difference in degree of flow for both techniques when comparing controls and all degrees of torsion combined (p < .006, Mann-Whitney test), but significance was achieved at lesser degrees of torsion with the velocity technique than with the energy technique (180 degrees and 360 degrees, respectively, Wilcoxon test). CONCLUSION: Color Doppler energy sonography was not significantly more sensitive than color Doppler velocity sonography for the diagnosis of spermatic cord torsion in this model. Complete occlusion of arterial inflow occurred at 450-540 degrees of torsion.

Allelic differences in the VHOx-1 gene explain the absence of a B cell clonal dominance in the primary response of C57BL/6 mice to phthalate.

A highly conserved Id (CRIXmp-1) associated with the murine (BALB/c) humoral immune response to the hapten phthalate (Xmp) is conspicuously absent in C57BL/6 mice. The absence of this Id in C57BL/6 mice is shown here to be due to the absence of the appropriate VH gene (VHOx-1) usage in the Xmp response. To determine whether the failure to utilize this VH was due to an active suppression or to the lack of the requisite VH gene in the available repertoire, VHOx-1 gene-specific primers were used to amplify the germ-line VHOx-1 gene from genomic DNA from BALB/c and C57BL/6 mice. The germ-line coding sequence of the C57BL/6 allele of the VHOx-1 gene is 99% similar to the germ-line coding sequence of the BALB/c allele. Amplification of cDNA made from splenic RNA from C57BL/6 mice confirmed that this gene is expressed. There are four nucleotide differences that lead to three amino acid changes in the predicted protein sequence. Each change is either in or immediately adjacent to a complementarity-determining region (CDR). Two of these changes are unique to the C57BL/6 allele and are not shared with CRIXmp-1-expressing strains. These two changes are predicted to alter the Xmp binding capabilities of the C57BL/6 allelic form of this VH gene, thereby explaining the absence of the Xmp-1 clonotype, which is dominant in the primary Xmp immune response of most other strains of mice.

Use of urea filter paper disc to detect urease activity in Enterobacteriaceae by multipoint replication techniques.

A new method of detecting urease activity in Enterobacteriaceae was developed. An 8.5 cm filter paper disc impregnated with 20% urea and 0.5% bromocresol purple was placed on the surface of a glucose fermentation plate after inoculation with a multipoint replicator and overnight incubation. This method was compared with the commercially prepared Mast urea agar (Multipoint) and Fuscoe's Urea Plate Medium. A total of 240 routine isolates of Enterobacteriaceae were tested for urease activity using the three methods. Sixty five isolates were positive by the three methods while 33 isolates gave differing results. The urea disc method was more sensitive for detecting urease activity in isolates of Klebsiella species, Morganella morganii, and Yersinia enterocolitica. It also overcame the problem associated with the other two media of diffusion of alkali end products through the medium.

Location of heme a on subunits I and II and copper on subunit II of cytochrome c oxidase.

A systemic study has been made of copper and heme a binding to subunits of beef heart cytochrome c oxidase. Copper and heme a were readily mobilized by ionic detergents, high ionic strengths, temperatures above 0 degrees C, thiol compounds, and gel-bound peroxides and free radicals when the subunits of the oxidase were dissociated from one another during polyacrylamide gel electrophoresis. Most subunits showed some affinity for heme a and copper under these conditions. However, in the presence of specific mixtures of ionic and nonionic detergents (e.g. 0.1% sodium dodecyl sulfate, 0.025% Triton X-100) at temperatures below 0 degrees C and in buffers of low ionic strength using 10 to 12% polyacrylamide gels preelectrophoresed for 3 days with thioglycolate, about 90% of the Cu was found on subunit II (Mr = 24,100), and heme a was found in equal amounts of subunits I (Mr = 35,800) and II. The oxidized-reduced and reduced-CO absorption spectra of these subunits resembled those of cytochrome c oxidase. It appears probable that in the native enzyme, subunit I contains heme a and subunit II contains copper and heme a. A relationship of mammalian cytochrome c oxidase to the two-subunit microbial cytochrome oxidase systems appears to exist.

Thermodynamic and EPR characteristics of a HiPIP-type iron-sulfur center in the succinate dehydrogenase of the respiratory chain.

In addition to the two species of ferredoxin-type iron-sulfur centers (Centers S-1 and S-2), a third iron-sulfur center (Center S-3), which is paramagnetic in the oxidezed state analogous to the bacterial high potential iron-sulfur protein, has bwen detected in the reconstitutively active soluble succinate dehydrogenase preparation. Midpoint potential (at pH 7.4) of Center S-3 determined in a particulate succinate-cytochrome c reductase is +60 +/- 15 mV. In soluble form, Center S-3 becomes extremely labile towards oxygen or ferricyanide plus phenazine methosulfate similar to reconstitutive activity of the dehydrogenase. Thus, even freshly prepared reconstitutively active enzyme preparations show EPR spectra of Center S-3 which correspond approximately to 0.5 eq per flavin; in particulate preparations this component was found in a 1:1 ratio to flavin. All reconstitutively inactive dehydrogenase preparations that Center S-3 is an innate constituent of succinate dehydrogenase and plays an important role in mediating electrons from the flavoprotein subunit to most probably ubiquinone and then to the cytochrome chain.