Estimation of the minimum mRNA splicing error rate in vertebrates.

The majority of protein coding genes in vertebrates contain several introns that are removed by the mRNA splicing machinery. Errors during splicing can generate aberrant transcripts and degrade the transmission of genetic information thus contributing to genomic instability and disease. However, estimating the error rate of constitutive splicing is complicated by the process of alternative splicing which can generate multiple alternative transcripts per locus and is particularly active in humans. In order to estimate the error frequency of constitutive mRNA splicing and avoid bias by alternative splicing we have characterized the frequency of splice variants at three loci, HPRT, POLB, and TRPV1 in multiple tissues of six vertebrate species. Our analysis revealed that the frequency of splice variants varied widely among loci, tissues, and species. However, the lowest observed frequency is quite constant among loci and approximately 0.1% aberrant transcripts per intron. Arguably this reflects the "irreducible" error rate of splicing, which consists primarily of the combination of replication errors by RNA polymerase II in splice consensus sequences and spliceosome errors in correctly pairing exons.

Primary renal lymphoma presenting with chronic low-grade fever.

Primary renal lymphoma (PRL) is a rare form of extranodal non-Hodgkin's lymphoma often diagnosed and treated by oncologists and urologists. Pathophysiological and clinical data on PRL are sparse, but the limited reported experience suggests the disease usually has an ominous outcome. As in other renal tumors, comprehensive radiological investigations have a central role in the recognition and final diagnosis of PRL. We describe the presenting features and clinical course of an elderly woman who was found to have PRL after evaluation for persistent low-grade fever. Diagnostic and therapeutic caveats are discussed on the basis of a critical literature review of case reports and descriptions of small series of patients.

Spontaneous frequency of exon skipping in the human HPRT gene.

In order to elucidate the mechanisms of mRNA splicing fidelity and the mutagenic potential of aberrant mis-spliced transcripts we have investigated the frequency of spontaneous exon skipping in the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in well characterized human primary fibroblasts isolated from two different individuals. In these cells, coexisting with the WT species, we also detected three aberrant HPRT transcripts missing exon IV, VII, or VIII. We were unable to detect transcripts missing exon II, III, V, or VI. Significantly, all the exons affected by skipping do not generate new stop codons more than 50 nucleotides upstream from the 3' most exon-exon junction. Exon VIII was the most prone to skipping with a relative frequency to WT of 0.019+/-0.004 (approximately one aberrant transcript per 50 WT transcripts). Exon IV exhibited a relative frequency of skipping of 0.006+/-0.002 ((approximately one aberrant transcript per 150 WT transcripts) and exon VII exhibited a relative frequency of skipping of 0.003+/-0.002 ((approximately one aberrant transcript per 300 WT transcripts). These data demonstrate that aberrant transcripts with exon skipped are generated spontaneously in humans and some appear to persist in the cell.

Enzymatic properties of rat DNA polymerase beta mutants obtained by randomized mutagenesis.

We have used random sequence mutagenesis to generate mutants of DNA polymerase beta in an effort to identify amino acid residues important for function, catalytic efficiency and fidelity of replication. A library containing 100 000 mutants at residues 274-278 in the N-helix of the thumb subdomain of the polymerase was constructed and screened for polymerase activity by genetic complementation. The genetic screen identified 4000 active pol beta mutants, 146 of which were sequenced. Each of the five positions mutagenized tolerated substitutions, but residues G274 and F278 were only found substituted in combination with mutations at other positions. The least conserved residue, D276, was replaced by a variety of amino acids and, therefore, does not appear to be essential for function. Steady-state kinetic analysis, however, demonstrated that D276 may be important for catalytic efficiency. Mutant D276E exhibited a 25-fold increase in catalytic efficiency over the wild-type enzyme but also a 25-fold increase in G:T misincorporation efficiency. We present a structural model that can account for the observations and we discuss the implications of this study for the question of enzyme optimization by natural selection.

Molecular analysis of T-lymphocyte HPRT- mutations in individuals exposed to ionizing radiation in Goiânia, Brazil.

We have characterized 54 HPRT- point mutations in T-lymphocytes from 17 individuals exposed to ionizing radiation of 137Cs in Goiania, Brazil and compared this spectrum to that of 30 HPRT- mutants from 9 unexposed Brazilian controls. The average internal exposure of the exposed group was 205 mCi, and the average external exposure was 1.7 Gy. The average HPRT- mutant frequency for the exposed group was 13.3 x 10(-5), approximately a 10-fold increase over the mutant frequency of the unexposed controls, which was 1.56 x 10(-5). The types of point mutations characterized included base substitutions, small deletions, frameshifts, insertions, complex mutations, and losses of exon sequences from the mRNA. The relative frequency of the different mutation types was similar in the two studied groups. However, in our study the distribution of events within the hprt coding sequence seemed to cluster at the same regions of the gene. These observations imply that the hprt gene does not present a homogeneous target to radiation mutagenesis, and perhaps this class of information may be used to detect radiation exposure in human populations.

Creating novel enzymes by applied molecular evolution.

Recent molecular techniques have made it feasible to simulate evolutionary processes and apply in vitro selection to evolve enzymes with novel properties that may have potential benefits for medical and industrial applications. The characterization of such mutants has also provided new insights into how molecular structure determines enzyme function.

Tolerance of different proteins for amino acid diversity.

Random mutagenesis of genes followed by positive genetic selection in bacteria requires that the variant molecules confer biological activity, and is thus the most demanding approach for generating new functionally active molecules. Furthermore, one can learn much about the protein in question by comparing the population of selected molecules to the library from which they were selected. Described here is a mathematical method designed to guide such comparisons. We use as examples the results of randomization-selection studies of four different proteins. There exists, in general, a positive correlation between the number of amino acid substitutions in a critical region of a protein and the likelihood of inactivation of that protein; a correlation long suspected, but developed here in detail. At this time, we are comparing regions in different proteins and our conclusions must be limited. However, the method presented can serve as a guideline for anticipating the yield of new active mutants in genetic complementation assays based on the extent of randomization.

Molecular analysis of mutations in mutator colorectal carcinoma cell lines.

The nature of mutations occurring in two colorectal carcinoma cell lines deficient in mismatch repair and displaying mutator phenotypes was determined. One of the lines (HCT116) exhibited a higher level of microsatellite instability than the second (DLD-1), although the rate of mutation at the selectable locus encoding the purine salvage enzyme hypoxanthine guanine phosphoribosyl transferase (HPRT) was equally elevated (about 350-450-fold relative to mismatch repair proficient cell lines). Transitions were the major class of mutations in the two mutator lines. In DLD-1 these mutations recurred at several sites that appeared to be hotspots. Frameshifts at a run of six guanine residues in the coding sequence for HPRT constituted 35% of mutations in HCT116. These frameshifts were highly unstable and reverted to wild type at high frequency. Larger deletions were also detected in HCT116. Although these deletions constituted a small proportion of mutations compared with the other types, our data suggest that the rate of deletion is elevated relative to mismatch repair proficient (hMLH1+) cell lines. These observations suggest that the gene(s) altered in DLD-1 may preferentially affect the repair of base mismatches while the alteration(s) in HCT116 may affect the repair of both mismatches and frameshifts.

Analysis of point mutations in the hprt gene of cancer patients treated with radioimmunoglobulin therapy.

The mutagenic impact of various environmental and therapeutic agents can now be directly assayed in humans by the T-lymphocyte cloning assay. We have previously reported that following radioimmunoglobulin therapy, cancer patients exhibited increased mutant frequency of the hprt locus and an increased yield of large intergenic deletions compared to unexposed controls. Here we report the results of the analysis of 26 independent hprt mutations in nine cancer patients who underwent radioimmunoglobulin therapy. The majority of mutations (52%) had lost exon sequences from the mRNA. The remaining mutations were 20% small deletions and frameshifts and 28% base substitutions. The type of mutations observed were similar to those seen in unexposed controls. The site distribution of the mutations, however, indicates that some sequence contexts may be more sensitive to radiation mutagenesis than others.

Mutator phenotypes in human colorectal carcinoma cell lines.

Recent studies have revealed that tumors in patients with hereditary nonpolyposis colon cancer are associated with high-frequency alterations of microsatellite sequences. To investigate the mechanisms and consequences of this form of genetic instability, we identified three colorectal carcinoma cell lines that express dinucleotide-repeat instability like that found in hereditary nonpolyposis colon cancer tumors and show increased rates of spontaneous mutation at selectable loci. However, the pattern of hypermutation in these cell lines differed significantly. In one line (HCT116), microsatellite mutations occurred at a remarkably high rate (approximately 10(-2) mutations per cell per generation), whereas this rate was considerably lower in the two other lines (DLD-1 and HCT15). The rate of mutation at the locus encoding hypoxanthine guanine phosphoribosyltransferase was substantially elevated (200- to 600-fold) in all three tumor cell lines, yet the types of mutations arising differed. A specific frame-shift hotspot accounted for 24% of hypoxanthine guanine phosphoribosyltransferase mutations in HCT116. The frequency of mutations at this site was reduced significantly in DLD-1 and HCT15 lines. These data suggest that the mutatw phenotypes in the colorectal carcinoma cell lines could be the consequence of mutator genes affecting different repair or error-avoidance pathways.

Strand bias in mutation involving 5-methylcytosine deamination in the human hprt gene.

Despite being generally under-represented in the genome, CpG sequences represent a disproportionately high fraction of sites involved in mutational events leading to human genetic disease. Cytosine within CpG dinucleotides is often modified to 5-methylcytosine. Deamination of 5-methylcytosine in situ yields a thymine, which being mispaired with guanine, is potentially mutagenic. Previous reports have indicated that most mutations recovered at these sites appear to originate on the non-transcribed strand as C-->T transitions. This trend may however, reflect the lack of detectable mutant phenotypes resulting from this transition at the complementary positions on the transcribed strand. To date, there has not been a good model system in which mutations can be recovered on both strands at the same CpG site. The human hprt gene has MeCpG sites contained within arginine codons for which mutations have been recovered on both strands. From an analysis of a database of hprt mutations, a statistically significant strand bias is observed in mutations recovered at CpG sites. We describe some models for the bias of mutation distribution observed at MeCpG sites in light of this and previous work are described.

Insertional inactivation of the tk locus in a human B lymphoblastoid cell line by a retroviral shuttle vector.

Insertional mutagenesis represents an inherent risk in retrovirally mediated gene therapy, but it may be a useful experimental strategy for identification and isolation of novel cellular loci. In this investigation we have established a model system using a heterozygous thymidine kinase (tk) marker locus in a human B lymphoblastoid cell line, and a M-MuLV based shuttle vector. The frequency of TK- mutants in cells carrying 1-2 proviruses per genome is approximately 2 x 10(-5), a 5-fold increase as compared to an uninfected control population. Southern analysis of a set of 13 retrovirus infected TK- mutants revealed a predominance of rearrangements among those mutants which had not undergone loss of heterozygosity. No consistent relationship was found to exist between the occurrence of a rearrangement and tk gene expression as detected by northern analysis. The mechanisms of retroviral shuttle vector insertional mutagenesis were characterized in more detail by focusing on a single TK- mutant, T2. The single proviral insert in T2 was found to lie within tk intron 2, in parallel orientation to the direction of tk transcription. DNA sequence analysis of tk cDNA revealed the presence of an aberrantly spliced product from which exon 4 is excluded. Aberrant splicing could sufficiently account for the low level of functional tk transcript and thus the TK- phenotype in T2, although potential contributions from other mechanisms cannot be excluded.

Coamplification of hprt cDNA and gamma T-cell receptor sequences from 6-thioguanine resistant human T-lymphocytes.

The nature of mutation at the HPRT locus in human T-lymphocytes in vivo is currently a subject of considerable interest. Determination of clonality in individual mutant T-lymphocytes is essential for the proper interpretation. This requires the molecular analysis of their respective T-cell receptors (TCR). We have developed a polymerase chain reaction (PCR)-based method for coamplification of hprt cDNA and the rearranged gamma T-cell receptor genes from crude cell lysates of individual 6-thioguanine resistant human T-lymphocytes. Following reverse transcription to produce hprt cDNA, the crude cell lysate is treated with proteinase K and subjected to a primary PCR with two sets of amplification primers, one specific for the hprt cDNA and the other for the rearranged gamma TCR gene. A secondary round of PCR, employing appropriate sets of nested amplification primers, are then used to produce sufficient quantities of DNA for both the sequencing and restriction fragment length analysis, of the hprt cDNA and gamma TCR gene respectively.

Investigation of the mutagenic specificity of X-rays using a retroviral shuttle vector in CHO cells.

In some biological systems ionizing radiation appears to induce large deletions and rearrangements, while in others point mutations predominate the mutational spectrum. Moreover, while the point mutations are often randomly distributed, some systems exhibit "hot spots." Retroviral shuttle vectors are particularly useful for investigating the basis of these differences since the genetic target can be conveniently analyzed in a variety of host backgrounds and genomic locations. We have studied the mutational specificity of X-rays in a Chinese hamster ovary cell line (CHO) containing a stably integrated retroviral shuttle vector, carrying the CHO aprt cDNA as the genetic target. Cells were irradiated with 7 Gy using a 180 kVp X-ray source. The predominant mutation (87% of all APRT mutants), as determined by Southern analysis, was the complete deletion of the shuttle vector construct. In addition, 23 APRT mutants, carrying an apparently intact shuttle vector, were characterized at the sequence level: 5 were transitions, 9 were transversions, 3 were small deletions or insertions, 4 were frameshifts, and 2 were small rearrangements. Although the type and the location of the point mutations characterized appeared largely random, small deletions, insertions, and frameshifts were frequently associated with direct sequence repeats.

Endogenous gene systems for the study of mutational specificity in mammalian cells.

Determination of the DNA sequence alterations produced by various mutagens is a prerequisite for understanding mechanisms of mutagenesis. With recent technical advances that permit rapid isolation of mutant alleles, the mammalian genome has become accessible to this type of analysis. Here we discuss the growing data base on mutational specificity in mammalian cells, with an emphasis on the information obtained from studies of two endogeneous genetic loci, aprt and hprt.

Perspectives on UV light mutagenesis: investigation of the CHO aprt gene carried on a retroviral shuttle vector.

The extent to which the cellular processing of shuttle vector-carried genes reflects that of endogenous chromosomal loci has been a subject of considerable controversy. In order to address this issue, we have developed a retroviral-based shuttle vector carrying the Chinese hamster ovary (CHO) adenine phosphoribosyltransferase (aprt) gene stably integrated into the genome to be used for studying mutational specificity in mammalian cells. Initially, we have characterized a collection of UV-induced mutants in a CHO cell background. We have therefore been able to directly compare this shuttle vector data to that previously obtained for UV-induced mutation at the endogenous CHO (aprt) locus. Although some potential differences between the two spectra have been noted, there appears to be a remarkable similarity in the distribution and site specificity of UV-induced mutations. These similarities extend to extrachromosomal shuttle vectors as well and consolidate the role of shuttle vectors as powerful analytical tools for studying mechanisms of point mutagenesis in mammalian cells.