Complement genes C1r and C1s feature an intronless serine protease domain closely related to haptoglobin.

The exon-intron structure of the human complement C1s gene displays a striking similarity with that of the gene encoding haptoglobin, a peculiar transport protein distantly related to the serine proteases. While the protease regions of the serine zymogens are typically encoded by multiple exons, the protease domains of C1s and of its genetically linked and functionally interacting homolog C1r are encoded as intronless domains, not unlike a region of haptoglobin, which in fact is devoid of proteolytic activity. The close similarity of the C1s gene with haptoglobin includes the precise conservation of exon-intron junctions and it extends to upstream exons encoding the short repeats typical of several complement components, but found also in other functionally unrelated proteins. Additional evidence of the common ancestry of C1r, C1s and haptoglobin is the presence, within the protease domain, of a set of sequence markers that distinguish these three proteins from all known serine proteases. The finding of vertebrate serine protease genes with an uninterrupted protease-encoding exon supports the definition of a novel evolutionary branch of this gene family and rules out the hypothesis that regards this unusual exon as an irrelevant byproduct of the extravagant functional divergence of haptoglobin.

Rapid detection by fluorescent multiplex PCR of exon deletions and duplications in the C1 inhibitor gene of hereditary angioedema patients.

Hereditary angioedema (HAE) is due to a variety of defects in the C1 inhibitor gene (C1NH gene), including approximately 20% of partial deletions/duplications whose boundaries are usually within Alu repeats. To ensure complete molecular characterization of C1 inhibitor deficiencies a fluorescent multiplex assay was constructed to amplify simultaneously five exons of C1NH and an exon of the BRCA1 gene. PCR protocols were optimized for these amplicons (size range between 300 and 700 bp). Forward and reverse chimeric primers that carry strand-specific 5' tags of 16 nucleotides were used to ensure similar levels of PCR products for each amplicon in the multiplex. Data were analyzed by superposing fluorescent profiles of test and control DNA and by visually comparing the normalized peak levels of corresponding amplicons, rather than by calculating the ratios of peak areas. Tests on a collection of known defects, including five different Alu-mediated deletions and a partial duplication have validated this approach. In a study of 19 sporadic cases of HAE, of which four had failed to reveal mutations upon screening all exons by fluorescent chemical cleavage, three de novo deletions were diagnosed by using this multiplex PCR approach: a deletion of exon 4, a deletion of exons 5 and 6, and an apparently complete gene deletion. Besides being suitable for the initial DNA screening of the C1NH gene in HAE patients prior to screening for point mutations, this method can be easily adapted to complex genes for the screening of rearrangements.

Molecular cloning of human C1 inhibitor: sequence homologies with alpha 1-antitrypsin and other members of the serpins superfamily.

Genetic and acquired diseases in man show that the proteolytic activity of the complement component C1 is crucially regulated by C1 inhibitor (C1-INH), a plasma protein whose suspected relatedness to other serine proteinase inhibitors (serpins) contrasts with its atypically large size and high degree of glycosylation. Indeed we have found that the C1-INH polypeptide precursor synthesized in a cell-free system is a 64-kDa protein, hence it exceeds the length of the precursor forms of typical serpins. Seeking more conclusive sequence information and a probe for the structural locus, we isolated C1-INH cDNA clones from a library representing size-enriched human liver mRNA. Nucleotide sequence analysis of a clone covering the carboxyterminal half of C1-INH conclusively documents the relatedness of this protein with the serpins, and reveals 27% amino acid identity with alpha 1-antitrypsin.

Synthesis and antituberculosis activity of thiocarboxamide derivatives of Schiff bases.

Synthesis of some N-(4-thiocarboxamidobenzylidene)arylamines and N-(substituted benzylidene)-p-thiocarboxamidoanilines from various arylaldehydes and arylamines is described. Fourteen representative compounds were tested in vitro on the ground H37RV.

Thermal and ultraviolet light inactivation of Mycobacterium africanum.

Mycobacterium africanum Yaoundé and Rwanda were more heat-resistant than the tubercle bacilli. The ultraviolet susceptibilities of M. africanum strains were within the range usually found in the mycobacteria (e-1 doses), however marked differences were found in the intersept numbers of the Yaounde and the Dakar types.

Complete cDNA sequence of human complement Cls and close physical linkage of the homologous genes Cls and Clr.

Overlapping molecular clones encoding the complement subcomponent Cls were isolated from a human liver cDNA library. The nucleotide sequence reconstructed from these clones spans about 85% of the length of the liver Cls messenger RNAs, which occur in three distinct size classes around 3 kilobases in length. Comparisons with the sequence of Clr, the other enzymatic subcomponent of Cl, reveal 40% amino acid identity and conservation of all the cysteine residues. Beside the serine protease domain, the following sequence motifs, previously described in Clr, were also found in Cls: (a) two repeats of the type found in the Ba fragment of complement factor B and in several other complement but also noncomplement proteins, (b) a cysteine-rich segment homologous to the repeats of epidermal growth factor precursor, and (c) a duplicated segment found only in Clr and Cls. Differences in each of these structural motifs provide significant clues for the interpretation of the functional divergence of these interacting serine protease zymogens. Hybridizations of Clr and Cls probes to restriction endonuclease fragments of genomic DNA demonstrate close physical linkage of the corresponding genes. The implications of this finding are discussed with respect to the evolution of Clr and Cls after their origin by tandem gene duplication and to the previously observed combined hereditary deficiencies of Clr and Cls.

Sequence heterogeneity of murine complementary DNA clones related to the C4 and C4-Slp isoforms of the fourth complement component.

Two classes of mRNA encoding the murine C4 protein were identified by sequence analysis of clones isolated from a liver complementary DNA library. The divergence found within a 357 base pair sequence available for comparison is limited to five nucleotide replacements located in the region corresponding to the carboxy-terminal end of the C4d peptide fragment. One of the nucleotide substitutions influences the presence of a site for the Hind III restriction endonuclease. That this restriction site indeed discriminates the two non-allelic genes encoding the mouse C4 and C4-Slp isoforms has been demonstrated by Southern blot analysis and nucleotide sequencing at the genomic level. Circumstantial evidence supports the identification of the gene lacking the Hind III site in the region corresponding to the carboxy-terminal end of the C4d fragment as the one encoding the C4-Slp isotype.

Human complement C1r and C1s proteins and genes: studies with molecular probes.

The isolation of complementary DNA clones for both enzymic subcomponents of C1 has made it possible to derive their complete amino acid sequences and to verify and extend previous protein data. We review here recent advances in studies of the C1r and C1s proteins and of the corresponding genes, using molecular probes. The mosaic structure of these proteins has been compared to the exon-intron organization of the C1s gene. Surprisingly, the C1r and the C1s genes feature an intronless serine protease domain, at variance with all vertebrate serine proteases. Moreover, C1r and C1s are related in evolution to haptoglobin, a serine protease analog lacking enzymic activity. The C1r and C1s genes are closely linked in an unusual tail to tail orientation. These findings are discussed with regard to the apparently coordinate expression of these complement components and to the combined nature of most C1r and C1s deficiencies. We also discuss the implications of the successful production of C1r protein using recombinant DNA technology.

Molecular cloning, gene structure and expression profile of mouse C1 inhibitor.

The gene encoding C1 inhibitor, the major control element of activation of the classical pathway of complement and a major inhibitor of several plasma serine proteases, has been studied only in man, where deficiency of C1 inhibitor results in the dominantly transmitted disease hereditary angioedema. Full-length mouse C1 inhibitor cDNA and genomic clones were isolated and characterized as a first step towards the complete characterization of the pattern of C1 inhibitor expression and the production of an animal model of C1 inhibitor deficiency. Restriction-enzyme and sequence analyses of a full-length genomic clone demonstrated that the mouse gene has the same structure as the human homologue, but differs in size (9 kb versus 17 kb), mostly due to the presence of repetitive Alu elements in the human gene. Sequence comparisons in the promoter region indicate important similarities, i.e. the absence of a TATA box, the presence of an initiator sequence encompassing the transcription-start site and of a gamma-interferon-activated sequence (GAS) element at position -124 of the human sequence. A stretch of about 100 nucleotides in intron 1 reveals an unusually high degree of conservation for non-coding sequences and contains non-canonical but conserved tandemly arranged GAS elements at positions 369 and 388 of the human sequence. This finding supports the conclusions of functional studies on the human C1INH gene indicating a role of this region in modulation of transcription by interferons. The profile of C1 inhibitor expression in mouse liver, lung, heart, kidney, spleen and brain was determined by quantitative northern blot analysis.

Liver mRNA probes disclose two cytochrome P-450 genes duplicated in tandem with the complement C4 loci of the mouse H-2S region.

A search for uncharacterized genes of the S region of the murine H-2 major histocompatibility complex was undertaken; a series of cosmid clones previously aligned by overlap hybridizations were used as radiolabeled probes. Sequences hybridizing with liver poly(A)+ RNA were found within a cosmid covering a region 3' to the C4-Slp gene (the gene encoding the hemolytically inactive isoform of the fourth component of serum complement). Radiolabeled, short cDNA complementary to liver poly(A)+ RNA was used to establish the transcriptional polarity of the newly detected gene and to define fragments containing its 3' end. DNA sequence analyses and comparisons with porcine peptides established that the gene encodes the enzyme steroid 21-hydroxylase (EC 1.14.99.10), a cytochrome P-450 often referred to as P-450(C21), whose major site of expression is the adrenal gland. Two copies of the P-450(C21) gene, very similar yet distinguishable by restriction endonuclease analysis, were found individually associated with C4 and C4-Slp, genes that encode isoforms of mouse fourth component of complement. One of the P-450(C21) genes is coamplified with C4-Slp in H-2w7, a haplotype carrying a rare elongation of the S region. Comparisons with other members of the P-450 gene family show that the P-450(C21) genes encode peptides of extraordinary evolutionary conservation. The detection of a liver transcript of P-450(C21) raises the issue of the specific metabolic role of this enzyme in this organ and may have implications for the interpretation of human congenital adrenal hyperplasia.

Complete nucleotide sequence of the gene for human C1 inhibitor with an unusually high density of Alu elements.

The complete (17159 bp) nucleotide sequence of the gene for the human C1 inhibitor has been determined. The transcription initiation site was examined by primer extension using human liver mRNA, and the messenger 5'-end sequence was determined on clones obtained by the anchored polymerase chain reaction. The gene of this serpin molecule is split by seven introns, with junctions of phases zero and one. An outstanding feature of the intron sequences is the occurrence of 17 AluI repeats of all four ancestral subgroups, indicating that the gene has been invaded during consecutive waves of Alu amplification, including a recent one. These Alu repeats form the sites of deletion and insertion in several known lesions in the C1-inhibitor gene. There is no obvious promoter site of the TATA-box type at the 5' end of the gene, but instead it contains a region of potential H-DNA structure similar to that found upstream of the human c-myc gene.

Frequent de novo mutations and exon deletions in the C1inhibitor gene of patients with angioedema.

BACKGROUND: Cases of angioedema with no family history but with functionally low levels of C1 inhibitor and recurrent attacks are often observed. Clinical and biochemical data do not distinguish these cases from proven inherited forms of hereditary angioedema. OBJECTIVE: We sought to test the hypothesis of de novo mutations in patients affected by angioedema without a family history of the disease. METHODS: Among 137 independent kindreds followed for hereditary angioedema, 45 (32.8%) patients with early onset of the disease were registered as sporadic cases. Nineteen patients with unaffected parents were screened for point mutations and microdeletions-insertions by using fluorescence-assisted mismatch analysis. The biologic paternity of these patients was verified by determining their alleles at 4 microsatellite loci. Gross deletions were detected with Southern blot analysis. RESULTS: C1 inhibitor plasma levels measured in both parents of 24 sporadic patients were normal in all but 3 patients. Among the 19 patients studied at the DNA level, 9 de novo single nucleotide substitutions and 6 de novo microdeletions were found. De novo exon deletions were detected in 3 additional patients with Southern blot analysis. CONCLUSIONS: De novo C1inhibitor mutations and exon deletions account for at least 25% of all unrelated cases of angioedema. This finding has implications relevant to the genetic epidemiology and genetic counseling of this disease. The observation that 5 of the 9 de novo point mutations reproduce previously reported changes underlines the presence of multiple hot spots, two of which contain a CpG dinucleotide.

Recombinational biases in the rearranged C1-inhibitor genes of hereditary angioedema patients.

DNA structural changes responsible for hereditary angioedema were sought in the C1-inhibitor gene, which contains unusually dense clusters of Alu repeats in various orientations. Among patients belonging to 45 unrelated families, eight partial C1-inhibitor gene deletions and a partial duplication were found. Four deletions had one of the boundaries within the gene and the other in extragenic regions--in three cases 5' of the gene and in one case 3' of the gene. The boundaries of the partial duplication and of the remaining four deletions mapped instead within a few kilobases of exon 4. The same element--Alu 1--the first of three tandem Alu repeats preceding exon 4, contained one of the breakpoints of each of these five rearrangements. Moreover, these recombination breakpoints spread over the entire length of Alu 1, in contrast with the tight clustering observed near the 5' end of Alu sequences rearranged in other human genes. Thus, two uncommon recombinational biases are observed in the Alu rearrangements of hereditary angioedema patients; one promotes the occurrence of intragenic breakpoints in a single Alu repeat, and the other allows the breaks to be distributed over the entire Alu structure rather than within the hot spot of the left Alu monomer. A region of potential Z-DNA structure, located 1.7 kb upstream of Alu 1, may contribute to both peculiarities.