Fluorescence-based DNA minisequence analysis for detection of known single-base changes in genomic DNA.

We describe a rapid, automated method for direct detection of known single-base changes in genomic DNA. Fluorescence-based DNA minisequence analysis is employed in a template-dependent reaction which involves a single nucleotide extension of an oligonucleotide primer by the correct fluorescently-tagged dideoxynucleotide chain terminator. Detection following electrophoresis on denaturing acrylamide gels is facilitated by alkaline phosphatase treatment of reaction products after extension followed by isopropanol precipitation of the dye-tagged, single-base-extended primer to remove unincorporated deoxynucleotides. Fluorescence analysis of the incorporated dye tag reveals the identity of the template nucleotide immediately 3' to the primer site. This technique does not require radioactivity or biotinylated PCR product, relies on the incorporation of a single dideoxynucleotide terminator to extend the primer by one nucleotide and takes advantage of the sensitivity of fluorescent terminators developed for automated DNA sequence analysis. As a demonstration, we have applied the assay to human genomic DNA for detection of the sickle mutation in the beta-globin gene, and have also examined feasibility for simultaneous delineation using a multiplex-like strategy in a single gel-lane of some of the most common beta-thalassemia mutations in the Mediterranean basin.

Fluorescent approaches to diagnosis of Lesch-Nyhan syndrome and quantitative analysis of carrier status.

Lesch-Nyhan syndrome is an X-linked recessive disorder caused by molecular defects within the HPRT gene. Deletional forms of this syndrome, most of which are inherited, account for 15% of the cases. In addition, a large percentage of cases are due to de novo point mutations. We have used complementary fluorescence-based PCR assays to analyse disease-causing mutations in three unrelated families: (1) inheritance of dye-labelled PCR products of linked polymorphic loci mapping within and flanking the HPRT gene; (2) dye-labelled exon dosage analysis and (3) automated fluorescence-based DNA sequence analysis. Our results using fluorescent, dye-tagged PCR products show that inheritance of two polymorphic small tandem repeats, HPRTB [AGAT]n, mapping within intron 3 of the HPRT gene, and the CA-repeat at DXS294 can be used to establish linkage to the disease. In addition, we modified a previously described PCR protocol to use fluorescent dye-labelled oligoprimers and an ABI Gene Scanner in order to rapidly quantitate deletional forms of Lesch-Nyhan syndrome. Quantitative PCR analysis of individual exons followed by dosage analysis confirmed a deletion encompassing exon 9. A similar approach was used to confirm a previously described HPRT gene duplication involving exons 2 and 3. In this analysis, we co-amplified the HPRTB [AGAT]n and HUMARA [AGC]n repeats and confirmed increased exon dosage in carriers for the duplication. DNA sequence analysis remains the method of choice for delineating new disease-causing mutations, most of which are non-deletional forms of Lesch-Nyhan syndrome. We have also used a cycle-sequencing strategy employing dye-labelled dideoxy terminators and a laser-activated, fluorescence-emission DNA sequencer in order to define carrier status in 10 family members at risk for Lesch-Nyhan syndrome due to a splice donor mutation in intron 7. Our DNA sequence analyses corroborate small tandem repeat (STR) inheritance patterns in this family. Multiple fluorescence-based strategies should facilitate rapid diagnosis of the various Lesch-Nyhan disease-causing mutations.

The biochemical properties and transforming potential of human Wnt-2 are similar to Wnt-1.

Wnt-2 is a member of the Wnt gene family that includes the proto-oncogene Wnt-1 (formerly Int-1). Although the predicted protein product of the Wnt-2 gene has only 38% amino acid identity with Wnt-1, it exhibits significant conservation of structural properties, including a hydrophobic signal sequence and invariant spacing and conservation of 22 cysteine residues. We have sought to characterize the biological and biochemical properties of Wnt family members and here present a characterization of Wnt-2 protein and a comparison with Wnt-1. We demonstrate, using both CHO and AtT-20 cells transfected with human Wnt-2 cDNA, that Wnt-2 encodes a 33 kDa protein that is modified by N-linked glycosylation to a 35 kDa species. Secreted Wnt-2 protein was detected in the culture medium only after cells were treated with suramin indicating that, like Wnt-1 protein, Wnt-2 is tightly associated with the cell surface. Expression of Wnt-2 cDNA in the mammary epithelial cell line C57 mg results in loss of density-inhibited growth and induces a transformed phenotype in monolayer culture similar to the effects produced by Wnt-1. These results indicate that Wnt-2 shares several biochemical and biological characteristics with Wnt-1 and suggests that other Wnt family members, by virtue of conserved structural features, may also exhibit similar properties.

Expression of the TGF alpha integral membrane precursor induces transformation of NRK cells.

TGF alpha is one member of a family of soluble growth factors that are derived from integral-membrane precursors. The mature form of TGF alpha is released from its transmembrane precursor (proTGF alpha) by a protease that, in many tumor cells, is inefficient or limiting. We have previously established that, in the absence of processing, membrane-anchored proTGF alpha is biologically active and can interact with the EGF receptor on adjacent cells, thereby inducing the receptor's intrinsic tyrosine kinase activity. We further showed that this interaction leads to immediate downstream signal transduction as evidenced by Ca2+ mobilization. To extend these observations, and to investigate its transforming potential, we infected normal rat kidney (NRK) cells with retroviral expression vectors that encode mutated forms of proTGF alpha containing amino acid substitutions at the proteolytic cleavage sites. NRK cells harboring these mutant constructs do not secrete mature growth factor, but do express biologically active proTGF alpha on the cell surface as shown by their ability to induce the autophosphorylation of EGF receptor on neighboring A431 cells in co-culture. Expression of the mutant proTGF alpha molecules promoted the anchorage-independent growth of NRK cells in soft agar, and caused them to be tumorigenic when injected into nude mice. These results demonstrate that an interaction between EGF receptor and the integral membrane precursor to TGF alpha can provide a mitogenic stimulus that leads to transformation. They further suggest that the accumulation of proTGF alpha on the surface of some transformed cells has physiological relevance.

A truncated, secreted form of the epidermal growth factor receptor is encoded by an alternatively spliced transcript in normal rat tissue.

Two independent cDNA clones corresponding to a 2.7-kilobase (kb) epidermal growth factor receptor (EGF-R) mRNA were isolated from a rat liver cDNA library. Sequence analysis revealed 100% homology in the external domain when compared with the full-length rat EGF-R nucleotide sequence and 80 to 90% similarity relative to the human EGF-R. However, the 3'-terminal sequence of these clones did not match EGF-R or any other known sequence(s) and was distinct from the 3' end of the 2.8-kb mRNA, which encodes a truncated EGF-R in A431 cells. The deduced amino acid sequence revealed an open reading frame which is homologous to the external domain of the EGF-R but which terminates prior to the transmembrane region. Southern blot analysis of rat genomic DNA indicated that the 3'-terminal sequence of this transcript is derived from the EGF-R gene. Analysis of a genomic clone containing the 3' end of the 2.7-kb transcript revealed that this sequence is present as a discrete exon in the mid-region of the receptor gene in proximity to the exon encoding the transmembrane domain. Introduction of an expression vector containing the truncated EGF-R cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 95-kilodalton protein which was detected in conditioned media, by using antisera directed against the EGF-R. A similarly sized protein was also detected in the media of WB cells, a continuous, nontransformed line of rat hepatic epithelial cells. Northern (RNA blot) analysis established that the truncated receptor is encoded by a 2.7-kb transcript found in normal rat liver. Furthermore, Northern analysis of rat poly(A)+ RNA showed that the 2.7-kb EGF-R transcript is expressed at differing levels in various fetal and adult tissues. These data indicate that alternative splicing of the EGF-R primary transcript yields a 2.7-kb mRNA which codes for a truncated form of the receptor. This receptor is secreted by rat hepatic epithelial cells in culture, which suggests that it may be secreted by normal rat cells or tissues and perhaps serve an as yet unknown physiological function.

Characterization of the rat transforming growth factor alpha gene and identification of promoter sequences.

We have determined the complete nucleotide sequence of rat transforming growth factor alpha (TGF alpha) mRNA and characterized the six exons that encode this transcript. These six exons span approximately 85 kilobases of genomic DNA, with exons 1 to 3 separated by particularly large introns. What had previously been thought to represent a species-specific difference in the size of the TGF alpha precursor (proTGF alpha) is now shown to be due to microheterogeneity in the splicing of exons 2 and 3. This results from a tandem duplication of the acceptor CAG and gives rise to two alternate forms (159 and 160 amino acids) of the integral membrane precursor. Exon 6, which encodes the 3' untranslated region of TGF alpha mRNA, also encodes, on the opposite strand, a small (approximately 200-nucleotide) transcript whose sequence predicts an open reading frame of 51 amino acids. Expression of this latter transcript does not appear to be coregulated with that of TGF alpha mRNA. Primer extension and S1 nuclease analyses of authentic TGF alpha transcripts revealed two major and multiple minor 5' ends which span more than 200 base pairs of DNA in a G + C-rich region that lacks canonical CCAAT or TATA sequences. The 5' ends of six independently derived cDNAs localized to five different sites in this same region. Restriction fragments that overlap these transcription start sites and extend approximately 300 base pairs in the 5' direction faithfully promote transcription in vitro with HeLa cell nuclear extracts. In addition, they direct the expression of the bacterial chloramphenicol acetyltransferase gene in transient-transfection assays.

Regulation in Escherichia coli of the porin protein gene encoded by lambdoid bacteriophages.

Specialized lambda transducing phages carrying the cloned lc porin gene from the lambdoid bacteriophage PA-2, including various amounts of a sequence 5' to the start of transcription, were used to study the regulation of the porin gene. It was found that a cyclic AMP receptor protein consensus binding site 65 base pairs 5' to the start of transcription was required for catabolite repression of lc but was not sufficient for maximum expression under derepressing conditions. A sequence located more than 209 base pairs 5' to the start of transcription was necessary for maximum expression. By manipulating the copy number of the lc gene and the temperature and by measuring both the rate of synthesis of mRNA and the amount of Lc protein in the outer membrane, it was determined that the expression of lc is regulated primarily at the level of transcription and that expression is not autoregulated. Evidence is also presented that the silent phage porin gene nmpC of Escherichia coli K-12 is transcribed to the same extent as lc even though it does not give rise to a stable pool of mRNA. The structure of the 5' end of lc and nmpC is similar to that of ompF, and a model for transcriptional regulation is presented which may apply to all of these porin genes.

Structure of the lc and nmpC outer membrane porin protein genes of lambdoid bacteriophage.

The lc gene of the lambdoid bacteriophage PA-2 and the nmpC gene located on a defective lambdoid prophage in the 12-min region of the Escherichia coli K12 chromosome have been sequenced. The porin proteins encoded by these two genes were almost identical, with only 4 of the 365 residues of the precursor forms of the proteins being different. The Lc and NmpC proteins were strongly homologous to the OmpC, ompF, and PhoE proteins, with greater than 56% of the residues identical in each case. Sequencing of the region flanking the lc gene allowed precise positioning of this gene with respect to the rightward cos site of the phage and to sequences which are homologous between PA-2 and lambda. In wild-type strains of E. coli K12, the nmpC gene is not expressed and contains an IS5 insertion near the 3' end of the coding region. This insertion deletes 18 residues from the COOH terminus of NmpC protein and adds 8 residues from an open reading frame extending into IS5 sequence. Expression of this form of the gene in an expression vector plasmid demonstrated that this altered protein is still capable of being translocated to the outer membrane. Plasmid expression experiments using lc-nmpC hybrid genes show that it is the presence of the IS5 insertion which prevents expression of the porin in wild-type E. coli K12. In the nmpC mutant which expresses the protein, there has been a precise excision of the IS5 which regenerates a COOH terminus of NmpC protein which is identical to that of the Lc protein. Blot hybridization detected no mRNA transcripts from the wild-type nmpC gene, although transcripts were readily detected from the lc gene in PA-2 lysogens and from the nmpC mutant which has excised the IS5. This indicates that IS5 affects the production or stability of transcripts from the adjacent nmpC gene.