Showing papers on "Pseudogene published in 1999"

Preservation of Duplicate Genes by Complementary, Degenerative Mutations

Abstract: The origin of organismal complexity is generally thought to be tightly coupled to the evolution of new gene functions arising subsequent to gene duplication. Under the classical model for the evolution of duplicate genes, one member of the duplicated pair usually degenerates within a few million years by accumulating deleterious mutations, while the other duplicate retains the original function. This model further predicts that on rare occasions, one duplicate may acquire a new adaptive function, resulting in the preservation of both members of the pair, one with the new function and the other retaining the old. However, empirical data suggest that a much greater proportion of gene duplicates is preserved than predicted by the classical model. Here we present a new conceptual framework for understanding the evolution of duplicate genes that may help explain this conundrum. Focusing on the regulatory complexity of eukaryotic genes, we show how complementary degenerative mutations in different regulatory elements of duplicated genes can facilitate the preservation of both duplicates, thereby increasing long-term opportunities for the evolution of new gene functions. The duplication-degeneration-complementation (DDC) model predicts that (1) degenerative mutations in regulatory elements can increase rather than reduce the probability of duplicate gene preservation and (2) the usual mechanism of duplicate gene preservation is the partitioning of ancestral functions rather than the evolution of new functions. We present several examples (including analysis of a new engrailed gene in zebrafish) that appear to be consistent with the DDC model, and we suggest several analytical and experimental approaches for determining whether the complementary loss of gene subfunctions or the acquisition of novel functions are likely to be the primary mechanisms for the preservation of gene duplicates. For a newly duplicated paralog, survival depends on the outcome of the race between entropic decay and chance acquisition of an advantageous regulatory mutation. Sidow (1996, p. 717) On one hand, it may fix an advantageous allele giving it a slightly different, and selectable, function from its original copy. This initial fixation provides substantial protection against future fixation of null mutations, allowing additional mutations to accumulate that refine functional differentiation. Alternatively, a duplicate locus can instead first fix a null allele, becoming a pseudogene. Walsh (1995, p. 426) Duplicated genes persist only if mutations create new and essential protein functions, an event that is predicted to occur rarely. Nadeau and Sankoff (1997, p. 1259) Thus overall, with complex metazoans, the major mechanism for retention of ancient gene duplicates would appear to have been the acquisition of novel expression sites for developmental genes, with its accompanying opportunity for new gene roles underlying the progressive extension of development itself. Cooke et al. (1997, p. 362)

Neuronal expression of neural nitric oxide synthase (nNOS) protein is suppressed by an antisense RNA transcribed from an NOS pseudogene.

Abstract: Here, we show that a nitric oxide synthase (NOS) pseudogene is expressed in the CNS of the snail Lymnaea stagnalis. The pseudo-NOS transcript includes a region of significant antisense homology to a previously reported neuronal NOS (nNOS)-encoding mRNA. This suggested that the pseudo-NOS transcript acts as a natural antisense regulator of nNOS protein synthesis. In support of this, we show that both the nNOS-encoding and the pseudo-NOS transcripts are coexpressed in giant identified neurons (the cerebral giant cells) in the cerebral ganglion. Moreover, reverse transcription-PCR experiments on RNA isolated from the CNS establish that stable RNA-RNA duplex molecules do form between the two transcripts in vivo. Using an in vitro translation assay, we further demonstrate that the antisense region of the pseudogene transcript prevents the translation of nNOS protein from the nNOS-encoding mRNA. By analyzing NOS RNA and nNOS protein expression in two different identified neurons, we find that when both the nNOS-encoding and the pseudo-NOS transcripts are present in the same neuron, nNOS enzyme activity is substantially suppressed. Importantly, these results show that a natural antisense mechanism can mediate the translational control of nNOS expression in the Lymnaea CNS. Our findings also suggest that transcribed pseudogenes are not entirely without purpose and are a potential source of a new class of regulatory gene in the nervous system.

Human stearoyl-CoA desaturase: alternative transcripts generated from a single gene by usage of tandem polyadenylation sites.

Abstract: A critical step in the synthesis of unsaturated fatty acids is catalysed by stearoyl-CoA desaturase (Scd). To determine the regulation of human Scd, we characterized the gene and its transcripts. Screening a human keratinocyte cDNA library and analysis of 3'-RACE (rapid amplification of cDNA ends) products from various tissues yielded a 5.2 kb cDNA encoding a 359 amino acid protein with a calculated molecular mass of 41.5 kDa. Analysis of 3'-RACE products suggested that alternative usage of polyadenylation sites generates two transcripts of 3.9 and 5.2 kb, a result consistent with Northern analysis. Southern analysis demonstrated the existance of two SCD loci in the human genome. Chromosomal mapping localized one locus to chromosome 10, and the second locus to chromosome 17. Characterization of genomic clones isolated from chromosome-specific libraries revealed that only the locus on chromosome 10 contained introns. Sequence analysis of the intron-less locus displayed multiple nucleotide insertions and deletions, as well as in-frame stop codons. Reverse transcriptase-PCR analysis performed with primers specific to the intron-less locus failed to produce a PCR product from brain, liver and skin RNA, indicating that the locus on chromosome 17 is most likely a transcriptionally inactive, fully processed pseudogene. These results suggest strongly that there is one structural SCD gene in the human genome, and that it generates two transcripts by use of alternative polyadenyation sites. Although the primary sequence and intron-exon structure of SCD is phylogenetically conserved, divergence between rodent and human is seen in the number of SCD genes and in the generation of alternative transcripts, suggesting a species-specific component of SCD regulation and function.

Highly Sensitive and Specific Fluorescence Reverse Transcription-PCR Assay for the Pseudogene-free Detection of β-Actin Transcripts as Quantitative Reference

Abstract: The use of common reverse transcription (RT)-PCR reference target sequences can produce false-positive results by amplification of either contaminating DNA or processed pseudogenes. Furthermore, qualitative RT-PCR alone cannot distinguish between high- and poor-quality cDNA preparations, which again may be crucial for the interpretation of low-abundance transcripts. We have developed a highly sensitive quantitative RT-PCR for β-actin, using the TaqManTM chemistry. Through this technique, we were able to quantitatively detect 10 β-actin molecules per 100 ng of cDNA without coamplification of pseudogenes or genomic DNA. Thus, the presented method may be advantageous for the interpretation of quantitative RT-PCR results. PCR analysis may be difficult to interpret unless a reference target sequence is amplified in parallel. This control reaction is necessary to evaluate whether a sufficient amount of amplifiable material is present in the sample investigated. Thus, negative PCR can be defined as such only when amplification of a reference gene reveals a positive result. Amplifying a reference is especially important in RT-PCR because RNA can be degraded rapidly before or during cDNA synthesis. As reference sequences for RT-PCR, so called “housekeeping” genes are preferred because they are constitutionally expressed by all cell types. β-Actin is an attractive candidate for reference coamplification because it exhibits only minor intraindividual kinetic changes and is not primarily affected by any human disease (1). However, a major concern with β-actin and other commonly used references such as glyceraldehyde-3-phosphate dehydrogenase is that processed pseudogenes can be coamplified by primers that are originally restricted to cDNA. This problem is difficult to circumvent because sequence homologies of >90% exist between mRNA and the respective pseudogenes (2); thus, the pseudogenes usually cannot be distinguished in gel electrophoresis. Furthermore, as long as genomic DNA contamination cannot safely be excluded, DNA may be coamplified together with cDNA. Thus, positive analysis of …

Duplicated genes evolve independently after polyploid formation in cotton

Abstract: Of the many processes that generate gene duplications, polyploidy is unique in that entire genomes are duplicated. This process has been important in the evolution of many eukaryotic groups, and it occurs with high frequency in plants. Recent evidence suggests that polyploidization may be accompanied by rapid genomic changes, but the evolutionary fate of discrete loci recently doubled by polyploidy (homoeologues) has not been studied. Here we use locus-specific isolation techniques with comparative mapping to characterize the evolution of homoeologous loci in allopolyploid cotton (Gossypium hirsutum) and in species representing its diploid progenitors. We isolated and sequenced 16 loci from both genomes of the allopolyploid, from both progenitor diploid genomes and appropriate outgroups. Phylogenetic analysis of the resulting 73.5 kb of sequence data demonstrated that for all 16 loci (14.7 kb/genome), the topology expected from organismal history was recovered. In contrast to observations involving repetitive DNAs in cotton, there was no evidence of interaction among duplicated genes in the allopolyploid. Polyploidy was not accompanied by an obvious increase in mutations indicative of pseudogene formation. Additionally, differences in rates of divergence among homoeologues in polyploids and orthologues in diploids were indistinguishable across loci, with significant rate deviation restricted to two putative pseudogenes. Our results indicate that most duplicated genes in allopolyploid cotton evolve independently of each other and at the same rate as those of their diploid progenitors. These indications of genic stasis accompanying polyploidization provide a sharp contrast to recent examples of rapid genomic evolution in allopolyploids.

Genome degradation is an ongoing process in Rickettsia.

Abstract: To study reductive evolutionary processes in bacterial genomes, we examine sequences in the Rickettsia genomes which are unconstrained by selection and evolve as pseudogenes, one of which is the metK gene, which codes for AdoMet synthetase. Here, we sequenced the metK gene and three surrounding genes in eight different species of the genus Rickettsia. The metK gene was found to contain a high incidence of deletions in six lineages, while the three genes in its surroundings were functionally conserved in all eight lineages. A more drastic example of gene degradation was identified in the metK downstream region, which contained an open reading frame in Rickettsia felis. Remnants of this open reading frame could be reconstructed in five additional species by eliminating sites of frameshift mutations and termination codons. A detailed examination of the two reconstructed genes revealed that deletions strongly predominate over insertions and that there is a strong transition bias for point mutations which is coupled to an excess of GC-to-AT substitutions. Since the molecular evolution of these inactive genes should reflect the rates and patterns of neutral mutations, our results strongly suggest that there is a high spontaneous rate of deletions as well as a strong mutation bias toward AT pairs in the Rickettsia genomes. This may explain the low genomic G + C content (29%), the small genome size (1.1 Mb), and the high noncoding content (24%), as well as the presence of several pseudogenes in the Rickettsia prowazekii genome.

Identification of a novel cytokeratin 19 pseudogene that may interfere with reverse transcriptase-polymerase chain reaction assays used to detect micrometastatic tumor cells

Abstract: In many recent publications, it has been claimed that reverse transcriptase-polymerase chain reaction (RT-PCR) assays involving genes with tissue-restricted expression can be used for specific and sensitive detection of cancer cells in blood, bone marrow and lymph nodes. Many different target mRNAs have been evaluated for such purposes. One of the most extensively studied genes, CK19, is predominantly expressed in cells of epithelial origin and normally not at detectable levels in hematopoietic or lymphatic tissues. Based on previous reports on CK19 we wanted to establish a useful assay for detection of micrometastatic cells. RNA and DNA specimens extracted from peripheral blood nucleated cells of healthy volunteers, as well as cell lines positive and negative for CK19 expression, were used in nested RT-PCR assays. Using previously published primers, we found a novel pseudogene that shows a high degree of identity with the CK19 gene sequence, except for differences caused by 3 small deletions and a number of point mutations, resulting in termination codons and frameshifts. The gene has therefore no coding potential. Importantly, published primer sequences and reaction conditions used by several other groups to detect CK19 mRNA may have led to the amplification of this pseudogene. The data illustrate one of the problems that must be addressed in validating RT-PCR assays for micrometastasis detection, and it is suggested that previous work using CK19 as a marker should be reassessed in view of the present finding.

Patterns of nucleotide substitution in Drosophila and mammalian genomes

Abstract: To estimate patterns of molecular evolution of unconstrained DNA sequences, we used maximum parsimony to separate phylogenetic trees of a non-long terminal repeat retrotransposable element into either internal branches, representing mainly the constrained evolution of active lineages, or into terminal branches, representing mainly nonfunctional “dead-on-arrival” copies that are unconstrained by selection and evolve as pseudogenes. The pattern of nucleotide substitutions in unconstrained sequences is expected to be congruent with the pattern of point mutation. We examined the retrotransposon Helena in the Drosophila virilis species group (subgenus Drosophila) and the Drosophila melanogaster species subgroup (subgenus Sophophora). The patterns of point mutation are indistinguishable, suggesting considerable stability over evolutionary time (40–60 million years). The relative frequencies of different point mutations are unequal, but the “transition bias” results largely from an ≈2-fold excess of G⋅C to A⋅T substitutions. Spontaneous mutation is biased toward A⋅T base pairs, with an expected mutational equilibrium of ≈65% A + T (quite similar to that of long introns). These data also enable the first detailed comparison of patterns of point mutations in Drosophila and mammals. Although the patterns are different, all of the statistical significance comes from a much greater rate of G⋅C to A⋅T substitution in mammals, probably because of methylated cytosine “hotspots.” When the G⋅C to A⋅T substitutions are discounted, the remaining differences are considerably reduced and not statistically significant.

Mutational pattern of the nurse shark antigen receptor gene (NAR) is similar to that of mammalian Ig genes and to spontaneous mutations in evolution: the translesion synthesis model of somatic hypermutation.

Abstract: The pattern of somatic mutations of shark and frog Ig is distinct from somatic hypermutation of Ig in mammals in that there is a bias to mutate GC base pairs and a low frequency of mutations. Previous analysis of the new antigen receptor gene in nurse sharks (NAR), however, revealed no bias to mutate GC base pairs and the frequency of mutation was comparable to that of mammalian IgG. Here, we analyzed 1023 mutations in NAR and found no targeting of the mechanism to any particular nucleotide but did obtain strong evidence for a transition bias and for strand polarity. As seen for all species studied to date, the serine codon AGC/T in NAR was a mutational hotspot. The NAR mutational pattern is most similar to that of mammalian IgG and furthermore both are strikingly akin to mutations acquired during the neutral evolution of nuclear pseudogenes, suggesting that a similar mechanism is at work for both processes. In yeast, most spontaneous mutations are introduced by the translesion synthesis DNA polymerase z (REV3) and in various DNA repair-deficient backgrounds transitions were more often REV3-dependent than were transversions. Therefore, we propose a model of somatic hypermutation where DNA polymerase z is recruited to the Ig locus. An excess of DNA glycosylases in germinal center reactions may further enhance the mutation frequency by a REV3-dependent mutagenic process known as imbalanced base excision repair.

The variable genes and gene families of the mouse immunoglobulin κ locus

Abstract: In this report 118 mouse Vkappa genes are described which, together with the 22 Vkappa genes reported previously (T. Kirschbaum et al., Eur. J. Immunol. 1998. 28: 1458-1466) amount to 140 genes that had been cloned and sequenced in our laboratory. For 73 of them cDNAs are known, i. e. they have to be considered functional genes, although 10 genes of this group have 1-bp deviations from the canonical promoter, splice site or heptanucleotide recombination signal sequences. Twenty Vkappa genes have been defined as only potentially functional since they do not contain any defect, but no cDNAs have been found (yet) for them. Of the 140 Vkappa genes 47 are pseudogenes. There are indications that two to five Vkappa genes or pseudogenes exist in the kappa locus which we have not yet been able to clone. The 140 Vkappa genes and pseudogenes were assigned to 18 gene families, 4 of them being one-member families. This differs from previous enumerations of the families only by the combination of the Vkappa9 and Vkappa10 families and by the addition of the Vkappa dv gene as a new separate family. Sequence identity usually was 80% or above within the gene families and 55-80% between genes of different families. Many of the mouse Vkappa gene families show significant homologies to the human ones, indicating that in evolution Vkappa gene diversification predated the divergence of the primate and rodent clades.

Transcriptional analysis of the PTEN/MMAC1 pseudogene, ΨPTEN

Abstract: PTEN/MMAC1 is a recently characterized tumor suppressor. A pseudogene derived from the human PTEN/MMAC1 phosphatase, ΨPTEN, has been reported. Recent analysis of the pseudogene revealed conflicting results about the expression of ΨPTEN. In this study, we show that the PTEN/MMAC1 pseudogene is actively transcribed in all cells and tissues examined. In some cases, pseudogene transcripts were found to represent as much as 70% of the total PTEN/MMAC1 RNA. As ΨPTEN is transcribed, there is a potential for misinterpretation of PTEN/MMAC1 mutations when RT – PCR techniques are used, as well as potential for a ΨPTEN-encoded translation product. Although we were unable to detect a pseudogene protein product in the cell lines examined, a baculovirus produced GST pseudogene fusion protein exhibited phosphatase activity comparable to wild type. The results of this study, taken together, indicate the potential complication of PTEN/MMAC1 molecular analysis caused by the expression of ΨPTEN.

Identification of an Active Reverse Transcriptase Enzyme Encoded by a Human Endogenous HERV-K Retrovirus

Abstract: Of the numerous endogenous retroviral elements that are present in the human genome, the abundant HERV-K family is distinct because several members are transcriptionally active and coding for biologically active proteins. A detailed phylogeny of the HERV-K family based on the partial sequence of the reverse transcriptase (RT) gene revealed a high incidence of an intact RT open reading frame within the HML-2 subgroup of HERV-K elements. In this study, we report the cloning of six full-length HML-2 RT genes, of which five contain an uninterrupted open reading frame. The RT enzymes were expressed as glutathione S-transferase fusion proteins in Escherichia coli, and several HERV-K RT enzymes demonstrated polymerase as well as RNase H activity. Several biochemical properties of the RT polymerase were analyzed, including the template requirements and optimal reaction conditions (temperature, type of divalent cation). Inspection of the nucleotide sequence of the HERV-K RT genes demonstrated a mosaic structure, suggesting that a high level of genetic recombination has occurred in this virus family, which is a hallmark of replication by means of reverse transcription. The selective pressure to maintain the RT coding potential is illustrated by the sequence of a particular HERV-K isolate that contains three 1-nucleotide deletions within a small RT segment, thus maintaining the open reading frame. These combined results may suggest that these endogenous RT enzymes still have a biological function. It is possible that the RT activity was involved in the spread of this major class of retroelements by retrotransposition, and in fact it cannot be excluded that this retrovirus group is still mobile. The endogenous RT activity may also have been involved in the shaping of the human genome, e.g., by formation of pseudogenes.

Appendix to the report by R. Thiebe et al. Characteristics of the immunoglobulin Vκ genes, pseudogenes, relics and orphons in the mouse genome

Abstract: No abstracts

A testis-specific gene, TPTE, encodes a putative transmembrane tyrosine phosphatase and maps to the pericentromeric region of human chromosomes 21 and 13, and to chromosomes 15, 22, and Y.

Abstract: To contribute to the creation of a transcription map of human chromosome 21 (HC21) and to the identification of genes that may be involved in the pathogenesis of Down syndrome, exon trapping was performed from HC21-specific cosmids covering the entire chromosome. More than 700 exons have been identified to date. One such exon, hmc01a06, maps to YAC 831B6 which contains marker D21Z1 (alphoid repeats) and had previously been localized to the pericentromeric region of HC21. Northern-blot analysis revealed a 2.5-kb mRNA species strongly and exclusively expressed in the testis. We cloned the corresponding full-length cDNA, which encodes a predicted polypeptide of 551 amino acids with at least two potential transmembrane domains and a tyrosine phosphatase motif. The cDNA has sequence homology to chicken tensin, bovine auxilin and rat cyclin-G associated kinase (GAK). The entire polypeptide sequence also has significant homology to tumor suppressor PTEN/MMAC1 protein. We termed this novel gene/protein TPTE (transmembrane phosphatase with tensin homology). Polymerase chain reaction amplification, fluorescent in situ hybridization, Southern-blot and sequence analysis using monochromosomal somatic cell hybrids showed that this gene has highly homologous copies on HC13, 15, 22, and Y, in addition to its HC21 copy or copies. The estimated minimum number of copies of the TPTE gene in the haploid human genome is 7 in male and 6 in female. Zoo-blot analysis showed that TPTE is conserved between humans and other species. The biological function of the TPTE gene is presently unknown; however, its expression pattern, sequence homologies, and the presence of a potential tyrosine phosphatase domain suggest that it may be involved in signal transduction pathways of the endocrine or spermatogenetic function of the testis. It is also unknown whether all copies of TPTE are functional or whether some are pseudogenes. TPTE is, to our knowledge, the gene located closest to the human centromeric sequences.

Comparative sequence of human and mouse BAC clones from the mnd2 region of chromosome 2p13.

Abstract: The mouse mutation mnd2 causes an autosomal recessive disorder characterized by muscle atrophy and wasting (Jones et al. 1993). Homozygous mice exhibit unsteady gait, growth retardation, and juvenile lethality. The mnd2 mutation is located on mouse chromosome 6 in a region corresponding to human chromosome band 2p13.3. We localized the mnd2 gene previously to a nonrecombinant interval of 0.2 cM and generated a 400-kb P1 and BAC contig of the region (Weber et al. 1998). Eight genes were identified in the nonrecombinant region and seven were eliminated from further consideration as candidate genes because of their normal expression pattern and coding sequence. Characterization of the gene D6Mm5e was complicated by the very low abundance of the transcript. To determine the complete structure of D6Mm5e and to identify additional candidate genes for mnd2, we initiated large-scale genomic sequencing of the nonrecombinant region. Determining the complete gene content of the nonrecombinant interval in positional cloning is challenging because exons comprise a small fraction of genomic DNA and the available experimental methods for isolating exons are inefficient and labor intensive. Identification of genes that are expressed at a very low level is particularly difficult. Comparative large-scale sequence analysis is a newly feasible method for annotation of human genomic sequence. Comparison of 1196 orthologous mouse and human full-length mRNAs revealed an average of 85% nucleotide and protein sequence identity in the coding regions (Makalowski et al. 1996). Because coding sequences are among the most highly conserved in mammalian genomic DNA, they can be readily detected when the corresponding genomic sequences of human and mouse are compared (Hardison et al. l997). The effectiveness of this approach has been demonstrated in several recent studies (Galili et al. 1997; Gottlieb et al. 1997; Oeltjen et al. 1997; Ansari-Lari et al. 1998). We have combined exon amplification, cDNA isolation by RT–PCR, large-scale mouse genomic sequence analysis, and mouse/human comparative sequence analysis to identify genes in the mnd2 nonrecombinant region.

Coevolution of PERB11 (MIC) and HLA class I genes with HERV-16 and retroelements by extended genomic duplication.

Abstract: The recent availability of genomic sequence information for the class I region of the MHC has provided an opportunity to examine the genomic organization of HLA class I (HLAcI) and PERB11/MIC genes with a view to explaining their evolution from the perspective of extended genomic duplications rather than by simple gene duplications and/or gene conversion events. Analysis of genomic sequence from two regions of the MHC (the alpha- and beta-blocks) revealed that at least 6 PERB11 and 14 HLAcI genes, pseudogenes, and gene fragments are contained within extended duplicated segments. Each segment was searched for the presence of shared (paralogous) retroelements by RepeatMasker in order to use them as markers of evolution, genetic rearrangements, and evidence of segmental duplications. Shared Alu elements and other retroelements allowed the duplicated segments to be classified into five distinct groups (A to E) that could be further distilled down to an ancient preduplication segment containing a HLA and PERB11 gene, an endogenous retrovirus (HERV-16), and distinctive retroelements. The breakpoints within and between the different HLAcI segments were found mainly within the PERB11 and HLA genes, HERV-16, and other retroelements, suggesting that the latter have played a major role in duplication and indel events leading to the present organization of PERB11 and HLAcI genes. On the basis of the features contained within the segments, a coevolutionary model premised on tandem duplication of single and multipartite genomic segments is proposed. The model is used to explain the origins and genomic organization of retroelements, HERV-16, DNA transposons, PERB11, and HLAcI genes as distinct segmental combinations within the alpha- and beta-blocks of the human MHC.

Spinal muscular atrophy: untangling the knot?

Abstract: Spinal muscular atrophy (SMA), a clinically and genetically heterogeneous group of neuromuscular diseases, is a disorder of motor neurones characterised by degeneration of spinal cord anterior horn cells and muscular atrophy. SMA is an autosomal recessive disorder with a carrier frequency of about 1150. Three candidate genes, the survival motor neurone (SMN) gene, the neuronal inhibitory protein (NAIP) gene, and the p44 (subunit of basal transcription factor TFIIH) gene, have been considered as genes involved in this condition. The region spanning these genes has a complex organisation including duplications, repetitive sequences, truncated genes, and pseudogenes, which makes molecular analysis of this condition difficult. Although deletions have been found in the majority of SMA patients, a few microrearrangements (like duplications, missense mutations, microdeletions, and gene conversions) localised in the telomeric form of the SMN gene have also been reported. The function of the protein encoded by the SMN gene is still not fully understood but recent studies have indicated that it is found intracellularly in gems, novel nuclear structures. Its interaction with other proteins suggests a role in mRNA processing and metabolism. Whether the NAIP gene protein and other apoptosis associated proteins are directly involved in the initial stages of neurone degeneration and apoptosis, or acting downstream on the pathological pathway, has been difficult to determine. Further studies will be required to elucidate possible functional interactions between these proteins.

Deficiency of Human Complement Protein C4 Due to Identical Frameshift Mutations in the C4A and C4B Genes

Abstract: The complement protein C4, encoded by two genes (C4A and C4B) on chromosome 6p, is the most polymorphic among the MHC III gene products. We investigated the molecular basis of C4 deficiency in a Finnish woman with systemic lupus erythematosus. C4-specific mRNA was present at low concentrations in C4-deficient (C4D) patient fibroblasts, but no pro-C4 protein was detected. This defect in C4 expression was specific in that synthesis of two other complement proteins was normal. Analysis of genomic DNA showed that the proposita had both deleted and nonexpressed C4 genes. Each of her nonexpressed genes, a C4A null gene inherited from the mother, a C4A null gene, and a C4B null gene inherited from the father, all contained an identical 2-bp insertion (TC) after nucleotide 5880 in exon 29, providing the first confirmatory proof of the C4B pseudogene. This mutation has been previously found only in C4A null genes. Although the exon 29/30 junction is spliced accurately, this frameshift mutation generates a premature stop at codon 3 in exon 30. These truncated C4A and C4B gene products were confirmed through RT-PCR and sequence analysis. Among the possible genetic mechanisms that produce identical mutations is both genes, the most likely is a mutation in C4A followed by a gene conversion to generate the mutated C4B allele.