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At least 289 records · Page 16Linked to original sources
PubMed1995

Age-dependent carriage of multiple Plasmodium falciparum merozoite surface antigen-2 alleles in asymptomatic malaria infections.

Genetic diversity of the merozoite surface antigen-2 gene of the human malaria parasite Plasmodium falciparum has been analyzed in a Senegalese village where malaria is holoendemic. A cross-sectional survey of 65 residents was performed in 1992 during the high transmission season. Plasmodium falciparum was detected both by microscopy (77% positive samples) and DNA amplification using a single (29% or 38% positive samples, depending on the primers used) or nested polymerase chain reaction (PCR) (78% positive samples). The overlap between the positive nested PCR and microscopic examination was not complete. The PCR fragments were analyzed for size polymorphism on agarose gels, and were subsequently assigned to the major allelic families 3D7 or FC27 by hybridization with family-specific probes. Both allelic families were found, with a slightly higher prevalence for FC27. Chimeric alleles that failed to hybridize under stringent conditions to the reference probes were also observed. Some were typed using a novel PCR approach, using hybrid pairs of primers, consisting of a family-specific sense oligonucleotide combined with an antisense oligonucleotide specific for the other family. Combining typing techniques, 82% of the positive PCR results yielded more than one band. Both the overall number of fragments and the number of allelic types per carrier were markedly reduced around the age of 15 years. The number of DNA fragments decreased abruptly from an average of four per carrier before the age of 15 years to an average of two in individuals more than 15 years of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed2006

The application of laser microdissection to in planta gene expression profiling of the maize anthracnose stalk rot fungus Colletotrichum graminicola.

Laser microdissection (LM) offers a potential means for deep sampling of a fungal plant-pathogen transcriptome during the infection process using whole-genome DNA microarrays. The use of a fluorescent protein-expressing fungus can greatly facilitate the identification of fungal structures for LM sampling. However, fixation methods that preserve both tissue histology and protein fluorescence, and that also yield RNA of suitable quality for microarray applications, have not been reported. We developed a microwave-accelerated acetone fixation, paraffin-embedding method that fulfills these requirements and used it to prepare mature maize stalk tissues infected with an Anemonia majano cyan fluorescent protein-expressing isolate of the anthracnose stalk rot fungus Colletotrichum graminicola. We successfully used LM to isolate individual maize cells associated with C. graminicola hyphae at an early stage of infection. The LM-derived RNA, after two-round linear amplification, was of sufficient quality and quantity for global expression profiling using a fungal microarray. Comparing replicated LM samples representing an early stage of stalk cell infection with samples from in vitro-germinated conidia, we identified 437 and 370 C. graminicola genes showing significant up- or downregulation, respectively. We confirmed the differential expression of several representative transcripts by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and documented extensive overlap of this dataset with a PCR-subtraction library enriched for C. graminicola transcripts in planta. Our results demonstrate that LM is feasible for in planta pathogen expression profiling and can reveal clues about fungal genes involved in pathogenesis. The method in this report may be advantageous for visualizing a variety of cellular features that depend on a high degree of histochemical preservation and RNA integrity prior to LM.

Acetone↗
PubMed2004

Mutation screening of the mitochondrial genome using denaturing high-performance liquid chromatography.

Over 170 known mutations of the mitochondrial genome are responsible for disease. Due to the unique features of mitochondrial genetics, such patients are clinically diverse and difficult to diagnose. As pathogenic mitochondrial DNA (mtDNA) mutations are mostly heteroplasmic, denaturing high-performance liquid chromatography (DHPLC) could be used to detect these heteroplasmic species and therefore act as a rapid screening test for mtDNA mutations. The entire mitochondrial genome was amplified by PCR in 40 overlapping regions. In addition, known mtDNA mutants were constructed for each of these regions using a PCR-based site-directed mutagenesis approach. These mutants were used as positive controls and showed a detection limit of 3-10% heteroplasmy by DHPLC (depending on the specific mutation) compared to 40% for conventional sequencing. To further validate the screening test, mtDNA from 17 patients with seven different pathogenic mutations was used to compare mutation detection by DHPLC and conventional sequencing. DHPLC had a sensitivity of 88% compared to 82% for sequencing. This increased to 100% sensitivity for DHPLC when excluding the m.8993T>G mutation. DHPLC analysis is therefore a sensitive, rapid and cost-effective method to screen for mutations in the mitochondrial genome. The role of pyrosequencing in the quantitation of mutant load for known mtDNA mutations was highlighted using the m.3243A>G mutation as an illustrative example. Pyrosequencing analysis was able to discriminate samples containing as little as 5% heteroplasmy and proved to be an accurate and reproducible method for estimation of mutant load.

Base Sequence↗
PubMed2002

Physical and transcript map of the hereditary prostate cancer region at xq27.

We have recently mapped a locus for hereditary prostate cancer (termed HPCX) to the long arm of the X chromosome (Xq25-q27) through a genome-wide linkage study. Here we report the construction of an approximately 9-Mb sequence-ready bacterial clone contig map of Xq26.3-q27.3. The contig was constructed by screening BAC/PAC libraries with markers spaced at approximately 85-kb intervals. We identified overlapping clones by end-sequencing framework clones to generate 407 new sequence-tagged sites, followed by PCR verification of overlaps. Contig assembly was based on clone restriction fingerprinting and the landmark information. We identified a minimal overlap contig for genomic sequencing, which has yielded 7.7 Mb of finished sequence and 1.5 Mb of draft sequence. The transcriptional mapping effort localized 57 known and predicted genes by database searching, STS content mapping, and sequencing, followed by sequence annotation. These transcriptional units represent candidate genes for HPCX and multiple other hereditary diseases at Xq26.3-q27.3.

Chromosome Mapping↗
PubMed2006

A drastic reduction in DOF1 transcript levels does not affect C4-specific gene expression in maize.

The transcription factor DOF1 has been suggested to regulate photosynthetic gene expression in maize. By screening a RescueMu transposon-tagged mutant library, we identified a maize mutant with a transposon integration in the Dof1 gene 16 bp upstream of the transcription initiation site (TIS). Sequencing of the Dof1 promoter region revealed an unusual promoter structure missing any typical elements. Homozygous (ho) mutant lines were generated by selfing and subsequent PCR and DNA gel blot analyses. The transposon integration reduced Dof1 transcript levels to less than 20% compared to the wild-type and overlapping RT-PCR systems revealed that these transcripts were not initiated from the native transcription start site. Dof1 transcripts transiently accumulate in wild-type plants after illumination of darkened seedlings, but this accumulation cannot be observed in mutant lines. However, the time-course of transcript accumulation from the C(4)-specific phosphoenolpyruvate carboxylase (PEPC) gene, a possible target of DOF1, is not altered. Moreover, no impact on the steady-state levels of five additional transcripts involved in C(4)-metabolism can be observed. The contents of amino acids, glucose, and malate as well as the carbon to nitrogen ratio in the leaves remained unchanged when comparing wild-type and mutant plants. Our data question the importance of DOF1 in the control of photosynthetic gene expression in maize.

Carbon Dioxide↗
PubMed1995

Bile salt-dependent lipase transcripts in human fetal tissues.

In human fetal pancreas, we identified two cDNA transcripts of the bile salt-dependent lipase (BSDL) using reverse transcription followed by polymerase chain reaction (RT-PCR). The sequence of four overlapping segments obtained by RT-PCR matched the sequence of the 2.2 kb cDNA cloned from human adult pancreas (Reue et al. (1991) J. Lipid Res. 32, 267-276). A second RT-PCR product of approx. 1.1 kb was evidenced, the sequence of which corresponds to that of the BSDL-pseudogene transcript (Nilsson et al. (1993) Genomics, 17, 416-422). The short transcript is present in all tissues examined whereas the former one (2.2 kb) is either poorly (in liver and kidney) or not at all expressed in adult tissues, excepted in the pancreas. On the other hand, the 2.2 kb transcript specific of the BSDL gene was detected in all fetal tissues examined as early as the 6th week of gestation. Results also suggested that the fetal pancreas contains more 2.2 kb transcript than its adult counterpart. Therewith, BSDL was immuno-precipitated from fetal liver. The role of BSDL-gene expression during the fetal life is discussed.

Amino Acid Sequence↗
PubMed2000

Molecular diagnosis of Stickler syndrome: a COL2A1 stop codon mutation screening strategy that is not compromised by mutant mRNA instability.

We have developed a novel strategy for screening families with type 1 Stickler syndrome due to COL2A1 nonsense mutations, using a modified RNA-based protein truncation test. To overcome the problem of the unavailability of collagen II-producing cartilage cells, reverse transcription polymerase chain reaction (RT-PCR) was performed on the illegitimate transcripts of accessible cells (lymphoblasts and fibroblasts), which were pre-incubated with cycloheximide to prevent nonsense-mutation-induced mRNA decay. The five overlapping RT-PCR fragments covering the COL2A1 coding region were then transcribed and translated in vitro to identify smaller truncated protein products which result from a premature stop codon. This method was used to screen a 4-generation Stickler family and a protein truncating mutation was identified, which was present in all affected individuals. Targeted sequencing identified the mutation as a G(+1) to A substitution at the 5' splice donor site of intron 25, which led to the activation of a cryptic splice site 8-bp upstream causing aberrant mRNA splicing and a translational frameshift that introduced a premature stop codon. Mutant mRNA was undetectable without cycloheximide protection, demonstrating that the mutant mRNA was subjected to nonsense-mediated mRNA decay. As well as providing further evidence that type 1 Stickler syndrome results from COL2A1 premature stop codon mutations, this study suggests mutant mRNA instability leading to haploinsufficiency may also be an important, but previously unrecognized, molecular basis of Stickler syndrome. This rapid new test for COL2A1 nonsense mutations is of particular clinical importance to Stickler syndrome families, where the identification of individuals who are at risk of this potentially preventable form of blindness will allow them to undergo regular ophthalmological surveillance and preventative or early ameliorative treatment.

Abnormalities, Multiple↗
PubMed2005

A strategy for obtaining near full-length HCV cDNA clones (assemblicons) by assembly PCR.

Hepatitis C virus (HCV) genotype is a predictor of response, and guides the duration of antiviral therapy. However, with the exception of HCV genotype 1a, 1b and 2a, a limited number of clones from other genotypes exist. Here we report the optimization of long RT-PCR to generate three overlapping amplicons that span the near full length HCV genome from a panel of HCV genotypes (1a, 1b, 2a, 2b, 3a, 4a, 5a). Assembly-PCR (As-PCR) was used to construct near full-length cDNA clones (assemblicons) for each genotype. The optimization of the long RT-PCR on genotype 1a and 1b indicated that QIAamp Viral RNA kit (Qiagen, UK), Expand RT and Expand Long Template PCR system (Roche, UK), were the most efficient in producing the requisite three overlapping amplicons and assemblicons for each genotype. The genotype of each assemblicon was confirmed. Assemblicon generation was only possible when the overlapping amplicons were biotinylated. As-PCR obviated the need for time consuming ligations and cloning. The use of three overlapping amplicons in the construction of HCV assemblicons minimised the chimeric nature of the resultant clone. As-PCR may prove a methodological avenue through which a larger panel of consensus HCV clones could be made available for HCV in vitro investigation.

DNA, Complementary↗
PubMed2002

Mechanisms of inactivation of hepatitis a virus by chlorine.

The study was intended to investigate the feasibility of reverse transcription-PCR (RT-PCR) for evaluation of the efficacy of inactivation of viruses in water and to elucidate the mechanisms of inactivation of hepatitis A virus (HAV) by chlorine. Cell culture, enzyme-linked immunosorbent assay, and long-overlap RT-PCR were used to detect the infectivity, antigenicity, and entire genome of HAV inactivated or destroyed by chlorine. The cell culture results revealed the complete inactivation of infectivity after 30 min of exposure to 10 or 20 mg of chlorine per liter and the highest level of sensitivity in the 5' nontranslated regions (5'NTR), inactivation of which took as much time as the inactivation of infectivity of HAV by chlorine. However, antigenicity was not completely destroyed under these conditions. Some fractions in the coding region were resistant to chlorine. To determine the specific region of the 5'NTR lost, three segments of primers were redesigned to monitor the region from bp 1 to 1023 across the entire genome. It was shown that the sequence from bp 1 to 671 was the region most sensitive to chlorine. The results suggested that the inactivation of HAV by chlorine was due to the loss of the 5'NTR. It is believed that PCR can be used to assess the efficacy of disinfection of HAV by chlorine as well as to research the mechanisms of inactivation of viruses by disinfectants.

Cell Culture Techniques↗
PubMed2002

Identification and characterization of 3 novel genital human papillomaviruses by overlapping polymerase chain reaction: candHPV89, candHPV90, and candHPV91.

Three novel human papillomaviruses (HPVs), candHPV89, candHPV90, and candHPV91, that were previously identified from short polymerase chain reaction (PCR) fragments AE6/CP6108, JC9710, and JC9813, respectively, were cloned and characterized from cervicovaginal cells by use of an overlapping PCR method. The complete nucleotide sequences of candHPV89 (8078 bp), candHPV90 (8033 bp), and candHPV91 (7966 bp) were determined by sequence walking. candHPV89 and candHPV91 were closely related to HPV83 and HPV7 and were placed in the HPV genome homology groups A3 and A8, respectively, by phylogenetic analyses. The genome of candHPV90 was most closely related to HPV71, although these HPV genomes do not seem to form a single lineage, because of the disproportionate divergence of the HPV71 L1 region. On the basis of phylogenetic analyses and available clinical data, these 3 novel HPV genomes appear to have a low oncogenic risk and expand the heterogeneity of HPVs detected in the lower genital tract.

Adolescent↗
PubMed1999

Role of TSG101 in uterine cervix cancer.

OBJECTIVE: TSG101 was first described as a possible tumor suppressor gene in breast cancer. To determine whether TSG101 might play a role in cervical carcinogenesis, we examined a panel of cervical cancer cell lines and primary tumor specimens for transcript abnormalities and mutations in TSG101. METHODS: Total RNA was derived from cell line cultures or primary tumor specimens. We performed nested reverse transcription polymerase chain reaction (RT-PCR) with eight overlapping primer sets, followed by single-strand conformational polymorphism (SSCP) analysis, to screen for mutations in the TSG101 open reading frame. Representative normal and shifted SSCP bands were sequenced. To identify abnormal-sized transcripts, we performed RT-PCR with primers flanking the open reading frame followed by gel electrophoresis. RESULTS: Mutational analysis was performed on cDNAs from 20 primary cervical tumors and 8 cervical carcinoma cell lines. Two polymorphisms were identified, neither of which resulted in an altered amino acid sequence. Transcript analysis was performed on a subset of 16 primary cervix tumors and 6 cervix carcinoma cell lines. The wild-type transcript (1228 bp) was the dominant transcript expressed in all samples. A transcript measuring 330 bp was detected in 5 of 6 cell lines and 11 of 16 primary tumor specimens. CONCLUSION: Our results suggest that mutations in TSG101 rarely occur in carcinomas of the uterine cervix. However, the presence of minor aberrant TSG101 transcripts is a common feature. The relationship between aberrant transcription and carcinogenesis should be further investigated.

Acid Anhydride Hydrolases↗
PubMed1996

Construction of an internal control to quantitate multiple porcine cytokine mRNAs by RT-PCR.

A multiple internal control was constructed to be used as an exogenously added control in reverse transcription polymerase chain reaction (RT-PCR) for pig cytokines. It consists of 5' and 3' primer sequences in the order of beta 2 microglobulin (beta 2-m), IL-1, IL-4, IL-6, IL-8, IL-2, IL-10, TNF-alpha, TNF-beta and IFN-gamma. Construction was accomplished by overlapping and extension PCR (OE-PCR) utilizing short oligonucleotides. The primers were designed to give two products of different sizes on co-amplification of control and target RNAs by RT-PCR in a single tube. This permits analysis of message for several cytokines using a single exogenously added competitive template. Incorporated endonuclease sites allow construct modification by oligonucleotide addition.

Animals↗
PubMed2006

A simplified PCR methodology for semiquantitatively analyzing dengue viruses.

The standard methodology for titrating dengue viruses, the plaque assay, is slow, time consuming and relatively expensive. Other methods require machinery that may not be routinely accessible to all researchers, particularly those in developing nations. We therefore sought to develop a rapid, simplified semiquantitative polymerase chain reaction (PCR) methodology based on the use of a template mimic. In particular, it was desired that the mimic should be applicable for use a DNA template to avoid the requirement for producing an in vitro RNA transcript. A 511 base pair fragment of the capsid-PrM junction of dengue serotype 4 was cloned into pGEM-T Easy vector and subjected to splicing overlap extension-PCR to generate a 160 base pair deletion. The deleted plasmid mimic competed competitively against the parent plasmid as well as the first strand cDNA of all four dengue viruses. The primers used are specific for the dengue virus, and no product was seen with first strand cDNA from a closely related flavivirus, Japanese encephalitis virus. Under the conditions used, accurate quantitation of the dengue viruses in the range of 10(3) to 10(6) pfu can be achieved in a single day, as opposed to the 7 days required for conventional plaque assay.

Capsid↗
PubMed2003

Generation of chicken single chain antibody variable fragments (scFv) that differentiate and neutralize infectious bursal disease virus (IBDV).

Phage-displayed recombinant antibody libraries derived from splenic mRNA of chickens immunized with an Australian strain of infectious bursal disease virus (IBDV) were constructed as single chain variable fragments (scFv) by either overlap extension polymerase chain reaction (PCR) or sequential ligation of the individual heavy (V(H)) and light (V(L)) chain variable gene segments. Sequential cloning of the individual V(H) and V(L) genes into a newly constructed pCANTAB-link vector containing the synthetic linker sequence (Gly(4)Ser)(3) was more efficient than cloning by overlap extension PCR, increasing the library size 500 fold. Eighteen IBDV specific antibodies with unique scFv sequences were identified after panning the library against the immunizing antigen. Eight of the clones contained an identical V(H) gene but unique V(L) genes. In ELISA analysis using a panel of Australian and overseas IBDV strains, one scFv antibody was able to detect all strains, whilst 3 others could discriminate between Australian and overseas strains, classical and variant strains and Australian field strains and vaccine strains. In addition, some scFvs showed significant neutralization titres in vitro. This report shows that generation of chicken antibodies in vitro by recombinant means has considerable potential for producing antibodies of diverse specificity and neutralizing capacity.

Amino Acid Sequence↗
PubMed1995

Refinement of the spinal muscular atrophy locus by genetic and physical mapping.

We report the mapping and characterization of 12 microsatellite markers including 11 novel markers. All markers were generated from overlapping YAC clones that span the spinal muscular atrophy (SMA) locus. PCR amplification of 32 overlapping YAC clones shows that 9 of the new markers (those set in italics) map to the interval between the two previous closest flanking markers (D5S629 and D5S557): cen-D5S6-D5S125-D5S435-D5S1407- D5S629-D5S1410-D5S1411/D5S1412-D5S1413- D5S1414-D5Z8-D5Z9-CATT1-D5Z10/D5Z6- D5S557-D5S1408-D5S1409-D5S637-D5S351-MA P1B-tel. Four of these new markers detect multiple loci in and out of the SMA gene region. Genetic analysis of recombinant SMA families indicates that D5S1413 is a new proximal flanking locus for the SMA gene. Interestingly, among the 40 physically mapped loci, the 14 multilocus markers map contiguously to a genomic region that overlaps, and perhaps helps define, the minimum genetic region encompassing the SMA gene(s).

Base Sequence↗
PubMed2003

Organization of the shrimp vitellogenin gene: evidence of multiple genes and tissue specific expression by the ovary and hepatopancreas.

Vitellogenin is the major egg yolk protein synthesized in female shrimp during gonad maturation. Although there are several reports for the cloning of vitellogenin complementary DNA (cDNA) in different crustaceans, little is known of the gene organization of this protein. This study reports the first cloning and characterization of a full-length gene encoding the vitellogenin precursor from the shrimp Metapenaeus ensis. By genomic DNA library screening, six different lambda clones were isolated using shrimp partial gene sequence as probe. Initial DNA sequence determination revealed that these clones are derived from different genes with coding sequence similar to other crustacean vitellogenins. Two of these clones were used for further analysis. One of the lambda clones (lambda 3.3) carries most of the coding sequence that correspond to the M. ensis vitellogenin gene (MeVg1) and the other clone (lambda 8.3) carries a smaller portion of the coding sequence of a different vitellogenin gene (MeVg2). The lambda 3.3 clone was chosen for further characterization. To clone the remaining 5' end upstream promoter region, 5' untranslated region and the remaining coding sequence of MeVg1, a polymerase chain reaction (PCR)-based gene walking approach was used. Subsequently, a PCR clone with overlapping sequence identical to the genomic clone was obtained and the organization of MeVg1 gene was constructed. The MeVg1 gene consists of 15 exons and 14 introns spanning approximately 10 kb. Several potential cleavage sites were identified from the deduced vitellogenin precursor. Cleaving of the precursor in these sites would result in the production of several vitellogenin subunits. To clone the cDNA for the vitellogenin, 5' and 3' rapid amplification of cDNA ends was performed using ovary cDNA of the shrimp. A 4.4 kb 5' cDNA clone and a 4 kb 3' end cDNA clone were isolated. The size of the reconstructed cDNA for M. ensis Vg is 7.97 kb and consists of the longest open reading frame of 7776 bp. Unlike the vitellogenin precursor of most insects and vertebrates, the deduced vitellogenin precursor lacks the polyserine domain important for receptor-mediated endocytosis. Phylogenetic analysis revealed a closer relationship of the MeVg1 with other crustacean vitellogenins but distantly related to other invertebrate and vertebrate vitellogenins. By reverse transcription-PCR, we have demonstrated that the shrimp MeVg1 gene is expressed only in the ovary and hepatopancreas while the MeVg2 gene is expressed exclusively in the hepatopancreas. In conclusion, the shrimp ovary also contribute significantly in the production of vitellogenin at transcription level and the gene organization of the shrimp protein may provide an insight in the evolution of this group of important proteins.

Amino Acid Sequence↗
PubMed1999

Construction and characterization of a humanized single chain Fv antibody fragment against the main immunogenic region of the acetylcholine receptor.

The single chain Fv fragment of mAb198 (scFv198) directed against the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR), can efficiently protect the AChR in muscle cell cultures against the destructive activity of human myasthenic autoantibodies. Humanization of the scFv198 antibody fragment should prove useful for therapeutic application by reducing its immunogenicity. Framework sequences from human immunoglobulins homologous to the rat scFv198 sequences were selected and a totally synthetic humanized scFv198 antibody fragment was constructed in vitro. Humanized VH and VL domains were synthesized using two overlapping sets of 225 bases long oligonucleotides overlap extension and polymerase chain reaction (PCR), then assembled into a full-length gene by overlap extension of single-stranded DNA (ssDNA) fragments and PCR. The initial humanized antibody fragment had a very low affinity for the AChR. Molecular modeling was then performed and four residues from the framework regions (FR) of the humanized VH domain were selected to be replaced by the corresponding amino acid from the rat sequence. Three mutants were constructed by overlap extension, using PCR. The humanized variant containing replacements at VH residues 27, 29, 30 and 71 showed very good recovery of AChR binding activity; its binding affinities for Torpedo or human AChR (K(D): 8.5 or 323 nM, respectively) being only four times lower than those of the parental scFv198 (K(D): 2 or 80 nM, respectively). This variant was able to protect the human AChR against the binding of anti-MIR mAb and anti-alpha autoantibodies from a myasthenic patient. It was also able to protect AChR against antigenic modulation induced by the anti-MIR mAb198.

Animals↗
PubMed2000

Stable amino-acid sequence of the mannose-6-phosphate/insulin-like growth-factor-II receptor in ovarian carcinomas with loss of heterozygosity and in breast-cancer cell lines.

The mannose-6-phosphate/insulin-like growth factor 2 receptor (Man-6-P/IGFII receptor) is involved in lysosomal enzyme sorting, IGFII degradation and pro-TGFbeta activation. Genetic alterations in hepatocarcinomas and a few breast cancers suggest that this receptor behaves as a tumor suppressor. Moreover, hypersecretion and Man-6-P-independent targeting of cathepsins in breast and ovarian carcinomas also suggest alterations in this receptor. We studied the Man-6-P/IGFII receptor gene in 8 ovarian carcinomas, and 4 breast- and ovarian-cancer cell lines. The results confirmed a frequent loss of heterozygosity (LOH) in the 6q27-qter region in 5 out of 8 ovarian carcinomas. We used 23 overlapping RT-PCR fragments to sequence the whole coding region of the Man-6-P/IGFII receptor. The 2491 amino-acid sequence of this receptor was perfectly conserved in 9 out of 10 of our samples, including MCF7 and MDA-MB231 cells and 5 ovarian carcinomas with LOH. This allowed us to rectify the 2 previously published sequences which differed in several bases, and to propose a consensus amino-acid sequence. The only amino-acid change (Thr --> Ala) was in BG1 ovarian-cancer cells, and was due to an A-to-G substitution on one allele at nucleotide 2561. We found no bi-allelic alterations in the 9 ovarian carcinomas, but 3 silent nucleotide substitutions leading to a lower cordon usage in 2 ovarian carcinomas with LOH. No mutation of the Man-6-P/IGFII receptor coding sequence was found in breast-cancer cell lines to explain the cathepsin-D hypersecretion and Man-6-P-independent trafficking described. We propose that, in breast and ovarian cancers, the frequent loss of one allele, associated with over-expression of some of its ligands, might be sufficient to saturate the receptor protein, displace the ligands to other sites, and consequently facilitate tumor progression.

Adult↗