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PubMed2003

Fugu ESTs: new resources for transcription analysis and genome annotation.

The draft Fugu rubripes genome was released in 2002, at which time relatively few cDNAs were available to aid in the annotation of genes. The data presented here describe the sequencing and analysis of 24,398 expressed sequence tags (ESTs) generated from 15 different adult and juvenile Fugu tissues, 74% of which matched protein database entries. Analysis of the EST data compared with the Fugu genome data predicts that approximately 10,116 gene tags have been generated, covering almost one-third of Fugu predicted genes. This represents a remarkable economy of effort. Comparison with the Washington University zebrafish EST assemblies indicates strong conservation within fish species, but significant differences remain. This potentially represents divergence of sequence in the 5' terminal exons and UTRs between these two fish species, although clearly, complete EST data sets are not available for either species. This project provides new Fugu resources, and the analysis adds significant weight to the argument that EST programs remain an essential resource for genome exploitation and annotation. This is particularly timely with the increasing availability of draft genome sequence from different organisms and the mounting emphasis on gene function and regulation.

Animals↗
PubMed1988

Proteolytic processing of pro-alpha and pro-beta precursors from human beta-hexosaminidase. Generation of the mature alpha and beta a beta b subunits.

There are two major isozymes of human lysosomal beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52), hexosaminidase A, alpha(beta a beta b), and hexosaminidase B, 2(beta a beta b). The alpha subunit contains a single polypeptide chain, while the beta subunit is composed of two nonidentical chains (beta a and beta b) derived from a common pro-beta precursor. The mature subunits, like those of most lysosomal enzymes, are produced through the proteolytic processing of propolypeptides once they enter the lysosome. In order to define the structure of the alpha and beta subunits generated in the lysosome, the alpha, beta a, and beta b polypeptides of hexosaminidase A and B were separated by a combination of molecular sieve and ion exchange high performance liquid chromatography, and amino-terminal sequences were determined. These were localized to the deduced amino acid sequences of previously isolated cDNAs coding for the prepro-alpha and beta polypeptides. From this analysis, the sites of hydrolysis generating the mature alpha, beta a, and beta b chains from hexosaminidase A and B could be determined. First, the signal peptide, required for processing of the pre-propolypeptides through the rough endoplasmic reticulum was predicted from the first in-frame Met residue on the cDNA. Second, amino acid sequencing defined the amino termini of the mature polypeptide chains and identified the pro-sequences removed from both the pro-alpha and pro-beta polypeptides. Third, an internal cleavage resulted in the removal of a tetrapeptide, Arg-Gln-Asn-Lys, and tripeptide, Arg-Gln-Asn, from the pro-beta chain of hexosaminidase A and B, respectively , to generate the beta b and beta a chains. This result localized the beta b and beta a chains to the amino-terminal and carboxyl-terminal halves of the pro-beta sequence, respectively. Finally, we previously reported minimal or no carboxyl-terminal processing of the pro-beta chain in the lysosome. On the other hand, we suggest that there is trimming at the carboxyl terminus of the pro-alpha chain based on comparison of molecular weights of deglycosylated alpha with the isolated beta b and beta a chains comprising the mature beta subunit with those predicted from the cDNA. Thus, in the lysosome the pro forms of hexosaminidase A and B undergo extensive proteolytic processing which, while specific in nature, has the appearance of removing easily accessible, nonessential domains, rather than contributing to biosynthetic maturation of function.

Amino Acid Sequence↗
PubMed1994

Human erythrocyte ankyrin, a cytoskeleton component, generates the p57 membrane proteinase which cleaves C3, the third component of complement.

Human erythrocytes express a p57 membrane serine proteinase which cleaves C3, the third component of complement. Amino acid analysis of the first 20 N-terminal residues of the purified p57 proteinase demonstrated 100% homology with residues 910-929 of erythrocyte ankyrin, a sequence localized in its Mr = 62 kDa fragment. Thus, we analyzed whether ankyrin could generate the p57 C3-cleaving activity. We demonstrate herein that: 1) anti-ankyrin antibodies react with purified p57 proteinase; 2) anti-p57 proteinase antibodies react with purified ankyrin but not with spectrin, another cytoskeleton component; 3) while purified ankyrin did not carry any detectable proteinase activity, limited proteolysis of ankyrin by immobilized chymotrypsin generated this C3-cleaving serine proteinase. Spectrin did not generate any C3-cleaving activity. This is the first demonstration that erythrocyte ankyrin could generate a proteinase activity, which in addition, cleaves specifically human C3.

Amino Acid Sequence↗
PubMed1998

The Human Genome Project: from mapping to sequencing.

Until recently, the "human genome" programs were mainly directed towards the development of maps to identify disease genes. The genetic map comprises about 8000 highly informative second generation markers of the microsatellite type. The density of markers is now sufficient to localize a gene for a monogenic disease with a precision of 1 to 2 million base pairs easily, and to define intervals which contain susceptibility genes for multifactorial disorders. A third generation map based on single nucleotide polymorphisms that can be genotyped using DNA chip technology is in progress. The physical map, based on sets of overlapping yeast artificial chromosomes ordered using sequence-tagged sites, covers over 90% of the genome. However, this physical map cannot serve as a support for sequencing because of the numerous rearrangements that occur in yeast artificial chromosomes. An international network of laboratories has mapped a set of more than 30,000 expressed sequences from cDNAs using whole genome radiation hybrids that enable integration of genes within existing maps. The human genome program is now progressively shifting to massive sequencing, although sequence ready maps are not available for the major part of the human genome. Similarly, our capacity to interpret the available genomic sequence remains limited.

Genetic Linkage↗
PubMed1989

Molecular characterization of a major autoantibody-associated cross-reactive idiotype in Sjogren's syndrome.

Primary Sjogren's syndrome is an autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands, producing associated dry eyes (keratoconjunctivitis sicca), dry mouth, and intermittently swollen salivary glands. A high proportion of the infiltrating B lymphocytes express surface and cytoplasmic Ig bearing a kappa-L chain-associated CRI defined by reactivity with the murine mAb, 17.109. To determine the structural basis for CRI expression in this disease, we generated CRI+ lymphoblastoid cell lines and a cDNA library from lymphocytes extracted from Sjogren's syndrome patients' salivary gland biopsy specimens. Nucleic acid sequence analyses of the mRNA of one such 17.109-CRI+ lymphoblastoid cell line (NOV) reveals the expressed kappa light chain variable region gene (V kappa gene) to be homologous to Humkv325, a conserved V kappa gene used at relatively high frequency in certain B cell malignancies. In addition, synthetic oligonucleotides, corresponding to the first and third frameworks and the second complementarity determining region of the Humkv325 gene, were used to identify and isolate clones from a cDNA library generated from SS salivary gland lymphocytes. Clones annealing specifically with one or more of these oligonucleotide probes contained kappa light chain cDNA. The sequences corresponding to the variable region of two clones (Taykv320 and Taykv306) were homologous to Humkv325. The V kappa genes of four other cDNA clones (Taykv322, Taykv310, Taykv308, and Taykv312) most likely were generated somatically from the rearranged Humkv325 gene through a limited number of nucleic acid base substitutions. Our results suggest that the high frequency of 17.109-CRI expression in Sjogren's syndrome patients results from a multiclonal expansion of B cells using Humkv325, and that the expressed Humkv325 may undergo somatic diversification in an apparent Ag-driven response.

Amino Acid Sequence↗
PubMed1997

Antigenic variation of core, NS3, and NS5 proteins among genotypes of hepatitis C virus.

Assays that detect antibody to hepatitis C virus (HCV) are used to screen blood donors and patients with hepatitis. Current enzyme-linked immunosorbent assay (ELISA)-based methods are invariably based upon antigens from expressed recombinant proteins or oligopeptides from HCV type 1. Some HCV antigens used in screening assays are coded by regions of the HCV genome that show extensive variability; therefore, HCV type 1-based assays may be less effective for the detection of antibody elicited by infection with other genotypes. In this study, we have measured antibody reactivity of sera from 110 hepatitis C patients infected with type 1b, 3a, or 4a to genotype-specific and cross-reactive epitopes present in recombinant proteins from HCV genotypes 1b (core, NS3, and NS5), 3a (NS3, NS5), and 4a (core, NS3), corresponding to those used in current third-generation screening ELISAs. By comparing the serological reactivities of sera to type-homologous and type-heterologous antigens, we detected a significant type-specific component to the reactivity to NS3 (61 to 77% of the total reactivity) and NS5 (60% of the total reactivity). Furthermore, despite the similarities in the amino acid sequences of the core antigens of type 1b and type 4a, we also found significantly greater reactivity to type-homologous antigens, with approximately 25% of reactivity being type specific. These findings are consistent with previous findings of fivefold weaker reactivity of sera from HCV type 2- and HCV type 3-infected blood donors in the currently used third-generation ELISAs and suggest that these assays are suboptimal for screening populations in which the predominant genotype is not type 1.

Amino Acid Sequence↗
PubMed1998

Sequence analysis of the mip gene of the soilborne pathogen Legionella longbeachae.

To understand the basis of pathogenesis by Legionella longbeachae serogroup 1, the importance of the Mip protein in this species was examined. Amino-terminal analysis of the purified, cloned L. longbeachae serogroup 1 ATCC 33462 Mip protein confirmed that the cloned gene protein was expressed and processed in an Escherichia coli background. DNA sequence analysis of plasmid pIMVS27, containing the entire L. longbeachae serogroup 1 mip gene, revealed a high degree of homology to the mip gene of Legionella pneumophila serogroup 1, 76% homology at the DNA level and 87% identity at the amino acid level. Primer extension analysis determined that the start site of transcription was the same for both species, with some differences observed for the -10 and -35 promoter regions. Primers designed from the mip gene sequence obtained for L. longbeachae serogroup 1 ATCC 33462 were used to amplify the mip genes from L. longbeachae serogroup 2 ATCC 33484 and an Australian clinical isolate of L. longbeachae serogroup 1 A5H5. The mip gene from A5H5 was 100% identical to the type strain sequence. The serogroup 2 strain of L. longbeachae differed by 2 base pairs in third-codon positions. Allelic exchange mutagenesis was used to generate an isogenic mip mutant in ATCC 33462 and strain A5H5. The ATCC mip mutant was unable to infect a strain of Acanthamoebae sp. both in liquid and in a potting mix coculture system, while the A5H5 mip mutant behaved in a manner siilar to that of L. pneumophila serogroup 1, i.e., it displayed a reduced capacity to infect and multiply within Acanthamoebae. To determine if this mutation resulted in reduced virulence in the guinea pig animal model, the A5H5 mip mutant and its parent strain were assessed for their abilities to establish an infection after aerosol exposure. Unlike the virulent parent strain, the mutant strain did not kill any animals under two different dose regimes. The data indicate that the Mip protein plays an important role in the intracellular life cycle of L. longbeachae serogroup 1 species and is required for full virulence.

Amino Acid Sequence↗
PubMed1992

Junctions between genes in the haptoglobin gene cluster of primates.

To investigate the nature of the recombination that generated the haptoglobin three-gene cluster in Old World primates, we sequenced the region between the second gene (HPR) and the third gene (HPP) in chimpanzees (15 kb), as well as the region 3' to the cluster in humans (14 kb). Comparison to the previously sequenced human haptoglobin (HP) and HPR genes showed that the junction point between HP and HPR in humans (junction 1) was not identical to the junction point between the HPR and HPP genes of the chimpanzee (junction 2). An Alu sequence was found at each junction, but both Alu sequences lacked short direct repeats of the flanking genomic DNA. The lack of direct repeats implies that both junction Alu sequences are the products of recombination between different Alu elements. In addition, other insertion and deletion events are clustered in the regions near the junction Alu sequences. The observation that Alu sequences define the junctions between genes in the haptoglobin gene cluster emphasizes the importance of Alu sequences in the evolution of multigene families.

Animals↗
PubMed2000

Relative roles of somatic and Darwinian evolution in shaping the antibody response.

The need for a highly specific system of recognition in immunity has resulted in the evolution of several somatic mechanisms such as V(D)J recombination, to diversify the repertoire of B cells. Therefore, repertoire diversification is the driving force for the cells that constitute the bulk of the response to unpredictable pathogens, the B2 naive B cells. Predictability of antigen, on the other hand, has played a major role in shaping the neonatal repertoire, in which evolution to recognize commonly encountered pathogens has driven the germline sequence of several VH segments that are used frequently in the neonatal repertoire. A third population, the memory B cell population, is generated to respond to a known pathogen, but predictability of the pathogen is not acquired until after a first exposure. Therefore, it is somatic evolution in germinal centers that drives the generation of high-affinity memory B cells.

Animals↗
PubMed1997

Treatment of autoimmune diseases through manipulation of antigen presentation.

In rheumatoid arthritis, HLA-DR alleles associated with elevated relative risk share a common sequence in the third hypervariable domain of the major histocompatibility complex class II molecule (MHC II). Immunization of mice with a peptide vaccine comprised of the appropriate MHC II sequence in adjuvant blocked the onset and reduced the relapse rate of experimental autoimmune encephalomyelitis (EAE). A phase I clinical trial testing a single injection of a third hypervariable domain peptide from the HLA-DRB1*0401 sequence in alum adjuvant showed that the vaccine is well tolerated and generates an anti-HLA-DR antibody response in approximately 25% of the treated patients. A phase II trial testing multiple boosts is in progress. In a more antigen-specific approach, solubilized MHC II molecules loaded with an autoantigenic peptide are injected intravenously to induce unresponsiveness by the binding of the T-cell receptor (TCR) in the absence of costimulation. Appropriate soluble MHC II:autoantigenic peptide complexes inhibit the recall antigen proliferative response of T clones or draining lymph node cells, and reduce the progression of EAE and experimental autoimmune myasthenia gravis (EAMG). A test of a soluble HLA-DR2:myelin basic protein (MBP) complex in multiple sclerosis is progressing in phase I.

Animals↗
PubMed1987

Linkage analysis using multiple Xq DNA polymorphisms in normal families, families with the fragile X syndrome, and other families with X linked conditions.

Multipoint linkage analysis was undertaken with eight Xq cloned DNA sequences which identify one or more restriction fragment length polymorphisms in 26 families. These families comprise seven phase known normal families with three or more males in the third generation, seven families segregating for haemophilia B, one large family with dyskeratosis congenita, and 11 families with the fragile X syndrome. Phase known meioses informative for three or more loci supported the order centromere--DXYS1--DXS107--DXS102, DXS51--F9--FRAXA--DXS15, DXS52, F8--Xqter in each group of families studied. One of the normal families was segregating for protan colour blindness and showed a phase known recombination which would support the order centromere--F9--DXS52--CBP--Xqter. With the exception of DXYS1, all of these sequences have been localised to Xq27----qter by in situ hybridisation or hybridisation to Xq fragment panels, and on this basis should lie within 20 cM of one another. No recombination was observed between the sequences localised to Xq28, namely DXS52, F8, and DXS15 (between DXS15 and DXS52 Z = 12.25 at theta = 0 with confidence limits of 0 to 5 cM). However, an excess of recombination was apparent in the region of FRAXA with maximal lod scores as follows: F9 versus FRAXA (Z = 2.05, theta = 0.19), DXS52 versus FRAXA (Z = 1.85, theta = 0.26), and DXS15 versus FRAXA (Z = 1.33, theta = 0.27). No consistent differences were observed in the frequency of recombination when families with the fragile X syndrome were compared with normal families or families segregating for other X linked conditions. These results are compared with other published work and support the conclusion that although measurable linkage exists between these flanking markers and FRAXA, the intervals as measured by the frequency of meiotic recombination will seriously limit their clinical usefulness.

Chromosome Mapping↗
PubMed1997

Trimeresurus stejnegeri snake venom plasminogen activator. Site-directed mutagenesis and molecular modeling.

The specific plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) is a serine proteinase presenting 23% sequence identity with the proteinase domain of tissue type plasminogen activator, and 63% with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom that does not activate plasminogen. TSV-PA contains six disulfide bonds and has been successfully overexpressed in Escherichia coli (Zhang, Y., Wisner, A., Xiong, Y. L., and Bon, C. (1995) J. Biol. Chem. 270, 10246-10255). To identify the functional domains of TSV-PA, we focused on three short peptide fragments of TSV-PA showing important sequence differences with batroxobin and other venom serine proteinases. Molecular modeling shows that these sequences are located in surface loop regions, one of which is next to the catalytic site. When these sequences were replaced in TSV-PA by the equivalent batroxobin residues none generated either fibrinogen-clotting or direct fibrinogenolytic activity. Two of the replacements had little effect in general and are not critical to the specificity of TSV-PA for plasminogen. Nevertheless, the third replacement, produced by the conversion of the sequence DDE 96a-98 to NVI, significantly increased the Km for some tripeptide chromogenic substrates and resulted in undetectable plasminogen activation, indicating the key role that the sequence plays in substrate recognition by the enzyme.

Amino Acid Sequence↗
PubMed1995

Promoter binding factors regulating cyclin B transcription in the sea urchin embryo.

Cyclin B is a key regulatory protein of the cell cycle, central to the control of the G2/M transition. In the developing sea urchin embryo, the cyclin B gene is transcriptionally regulated in concert with changing patterns of cell division. In an effort to understand the mechanism controlling cyclin B expression during development, we have conducted an analysis of the Strongylocentrotus purpuratus cyclin B gene promoter. DNase I foot-printing of the cyclin B upstream region revealed eight binding regions within 435 bp of the start of transcription; seven of these sites were within 215 bp. Found within these regions were consensus sequences for two CCAAT boxes, TATA, and E-boxes and sequences with some similarity to E2F and octamer binding motifs. Upstream sequences were functionally defined by generating cyclin B-CAT fusion genes, containing deletions and base specific mutations, and testing for relative levels of expression by gene transfer. Both CCAAT boxes were found to be essential for maximal levels of expression. A third binding site (PR7) with no recognizable consensus sequence was also found to act as a positive element. Our results suggest that protein binding to the E2F-like sequences may act to reduce expression. Protein binding was further characterized by gel mobility-shift and methylation interference. The CCAAT boxes were found to bind similar, if not identical, proteins. Sequence comparisons and methylation interference data indicate that the likely protein binding these CCAAT sequences is the characterized CCAAT-binding protein CP1. A probe containing site PR7 formed multiple gel shift complexes that, by methylation interference, appeared to be interrelated. One major complex was formed with an oligonucleotide containing the two E2F-like sequences. Protein binding to this probe was specific and required bases within the E2F-like sequences. Our results indicate that cyclin B is subject to positive and negative regulation, involving multiple factors that bind between -200 and -90 bp from the start of transcription.

Animals↗
PubMed2003

Hydrolysis of third-generation cephalosporins by class C beta-lactamases. Structures of a transition state analog of cefotoxamine in wild-type and extended spectrum enzymes.

Bacterial resistance to the third-generation cephalosporins is an issue of great concern in current antibiotic therapeutics. An important source of this resistance is from production of extended-spectrum (ES) beta-lactamases by bacteria. The Enterobacter cloacae GC1 enzyme is an example of a class C ES beta-lactamase. Unlike wild-type (WT) forms, such as the E. cloacae P99 and Citrobacter freundii enzymes, the ES GC1 beta-lactamase is able to rapidly hydrolyze third-generation cephalosporins such as cefotaxime and ceftazidime. To understand the basis for this ES activity, m-nitrophenyl 2-(2-aminothiazol-4-yl)-2-[(Z)-methoxyimino]acetylaminomethyl phosphonate has been synthesized and characterized. This phosphonate was designed to generate a transition state analog for turnover of cefotaxime. The crystal structures of complexes of the phosphonate with both ES GC1 and WT C. freundii GN346 beta-lactamases have been determined to high resolution (1.4-1.5 Angstroms). The serine-bound analog of the tetrahedral transition state for deacylation exhibits a very different binding geometry in each enzyme. In the WT beta-lactamase the cefotaxime-like side chain is crowded against the Omega loop and must protrude from the binding site with its methyloxime branch exposed. In the ES enzyme, a mutated Omega loop adopts an alternate conformation allowing the side chain to be much more buried. During the binding and turnover of the cefotaxime substrate by this ES enzyme, it is proposed that ligand-protein contacts and intra-ligand contacts are considerably relieved relative to WT, facilitating positioning and activation of the hydrolytic water molecule. The ES beta-lactamase is thus able to efficiently inactivate third-generation cephalosporins.

Amino Acid Sequence↗
PubMed1986

[A study of proliferative responses to host Ia antigens in fully allogeneic bone marrow chimeras in mice--sequential analysis of the reactivity and characterization of the cells involved in the response].

Irradiation bone marrow chimeras were established by reconstitution of lethally irradiated AKR mice with C57BL/10 marrow cells which had pretreated with anti-Thy-1 serum. Mixed lymphocyte reactions (MLR) were carried out serially to analyze developing reactivities of lymphocytes from such chimeras, [B10----AKR], against donor, host or third party antigens. Spleen cells of [B10----AKR] chimeras 8 weeks after reconstitution regularly showed proliferative responses when stimulated with spleen cells of the strain of the irradiated recipient (AKR). However, the responsiveness was consistently lower than that generated against third party antigens. The stimulator cells were Ia positive in both anti-recipient and anti-third party responses, and the responding splenocytes were of donor origin and showed Thy-1+, Ly-1+2-, and L3T4+ phenotypes, although a minor population of Ly-2+ and L3T4- T cells might be involved in anti-third party response. Further, we found that substantial proliferative responses to Ia antigens of the recipient strain and also to third party antigens are generated by the thymocytes obtained from the irradiation chimeras at an early stage following bone marrow reconstitution. Majority of the responding thymocytes had surface traits of PNA-, donor type Thy-1+, and L3T4+ in both anti-recipient and anti-third party MLRs. However, as for the Ly-2 antigen, Ly-2+ thymocytes seemed to be, at least partially, involved in anti-host responses. This capacity of thymus cells to mount a response to antigens of the recipient strain declined shortly thereafter. The spleen cells at the same time developed a more durable capability to exhibit anti-host reactivities. Awareness of the sequence of development of these cellular reactivities and capacities to respond to host and third party alloantigens may be crucial to understanding the underlying molecular mechanisms involved in generating a T cell repertoire one component of which is restricted to Ia antigens of the recipient strain.

Animals↗
PubMed2000

Molecular cloning and characterization of mouse CD97.

The EGF-TM7 family (CD97 and EMR1) is a group of class II seven-span transmembrane receptors predominantly expressed by cells of the immune system. Recently, we have identified CD55, a regulatory molecule of the complement cascade, as a cellular ligand of human CD97 (hCD97). In this study, the molecular properties of mouse CD97 (mCD97) are described. Like hCD97, mCD97 has an extended extracellular region with several epidermal growth factor-like (EGF) domains. Due to alternative RNA splicing, isoforms with three and four EGF domains exist, designated mCD97(EGF1,2,4) and mCD97(EGF1,2, 3,4) respectively. All EGF domains, except for the N-terminal one, possess a calcium-binding site. In a third isoform mCD97(EGF1,2,X,3, 4), a sequence of 45 amino acids was found between the second and third EGF domain that does not correspond to any known protein module. Using newly generated mCD97 mAb, we show that analogous to the blood expression pattern of hCD97, mCD97 can be found on lymphoid and myeloid cells. Adhesion of mouse erythrocytes and splenocytes to COS cells expressing mCD97(EGF1,2,4) or mCD97(EGF1,2, 3,4) could be blocked by mouse CD55 (mCD55) antibody, identifying mCD55 as a cellular ligand for mCD97. Consistent with the necessity of directly linked EGF domains for the integrity of the CD55-binding site on hCD97, no adhesion was detected to the largest mouse isoform mCD97(EGF1,2,X,3,4). Remarkably, we found that the interaction between CD97 and CD55 is phylogenetically restricted, as indicated by the selective adhesion of primate erythrocytes to hCD97 transfectants, and of mouse and rat erythrocytes to mCD97 transfectants respectively.

Amino Acid Sequence↗
PubMed2000

Novel p53 splice site mutations in three families with Li-Fraumeni syndrome.

Germline mutations in the p53 tumor suppressor gene predispose to a variety of cancers in families with Li-Fraumeni syndrome. Most germline p53 mutations observed to date cause amino acid substitutions in the protein's central sequence-specific DNA binding domain. Outside this conserved core region, however, we found novel alterations in sequences that regulate precursor mRNA splicing in three Li-Fraumeni syndrome families. Two splice site mutations affected the consensus sequence at the splice donor sites of introns 1 and 9, and produced unstable variant transcripts in normal cells. A third mutation at the splice acceptor site of intron 9 generated splicing at a cryptic acceptor site in intron 9. These splice site alterations emphasize the need to examine both noncoding and untranslated regions of the p53 gene for germline mutations in Li-Fraumeni syndrome families. Oncogene (2000) 19, 4230 - 4235

Base Sequence↗