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PubMed1993

Sorbitol dehydrogenase. Full-length cDNA sequencing reveals a mRNA coding for a protein containing an additional 42 amino acids at the N-terminal end.

A cDNA clone encoding rat sorbitol dehydrogenase (SDH) was isolated from a rat testis lambda ZAP II cDNA library. The full-length cDNA insert contained 2277 base pairs (bp), starting 182 bp upstream from an ATG codon where translation to the active enzyme SDH is presumed to be initiated. A second ATG codon, however, was found 126 bp upstream, aligned in the same reading frame as that of the active enzyme. Therefore, the coding sequence for SDH can be translated into an additional 42-amino-acid polypeptide linked to the N-terminal amino acid of the enzyme, generating a pre-sorbitol dehydrogenase. The sequence data indicate that the nucleotide environment around this ATG codon is more favorable towards it being the actual open reading frame (ORF) for a pre-SDH than the ATG codon preceding the nucleotide sequence for SDH. Since no known SDH starts with the additional 42 amino acids, it may be that post-translational removal of this polypeptide accompanies the release of the active enzyme. Next, the 3' untranslated region of the cDNA contained a non-coding 1021 bp downstream from the TAA stop codon. The latter sequence included three putative poly(A) signals: one at nucleotides 1362-1367, the second at nucleotides 1465-1470, and the third at nucleotides 2212-2217 [17 bp away from the poly(A) tail]. In addition to the above findings we also report a variance in one of the amino acids in the SDH cDNA sequence. This variance occurs at position 957-960, where threonine is coded for instead of aspartic acid; in the rat testis SDH cDNA, we find the sequence is ACG instead of GAC, as was reported for the rat liver SDH cDNA. Northern-blot hybridization analysis showed that SDH mRNA is a doublet, one band of 4 kb and the other of 2.3-2.4 kb, in both the rat liver and the rat lens, further confirming that the isolated SDH cDNA constituted a full-length cDNA.

Amino Acid Sequence↗

PubMed2002

Integrative recombination between adenovirus type 12 DNA and mammalian DNA in a cell-free system: joining by short sequence homologies.

A cell-free system was developed to investigate the mechanism of how junctions are formed between viral and cellular DNAs during adenoviral DNA integration into the hamster cell genome. Recombination between the segment of adenovirus type 12 (Ad12) DNA, that comprises sequence coordinates 20885-24053, subsequently termed PstI-D fragment and the hamster preinsertion DNA sequence p7 was studied in a cell-free system. The p7 DNA segment had served as viral DNA integration site in the Ad12-induced tumor CLAC1. Nuclear extracts initially from uninfected BHK21 hamster cells were fractionated by a series of chromatographic steps. DNAs of the in vitro generated recombinants were analyzed in detail. In the course of the recombination reaction, the two linear molecules were joined. The reaction took place between two short homologous sequences one of which was always at or very close to a DNA terminus, the second one could be several kilobase pairs remote from a DNA terminus. Apparently, the nucleotide sequence at the terminus of one recombining molecule determined the point of junction by searching for short homologies in the partner molecule. The recombination reaction was not conservative, the sequences in-between the short sequence homologies and one of the short sequence homologies were deleted in the in vitro recombinants. Two main criteria influenced the choice of interacting short sequence homologies: perfect homologies of 8-9 bp were most frequently found, they were preferred over more extended, but less perfect homologies. Comparing different short sequence homologies with similar stabilities, those combinations seemed to be chosen in the reaction which led to a minimal loss of nucleotides in the recombinants. The in vitro activity was found in nuclear extracts from both hamster and human cells. The activity was, hence, available for Ad12 DNA in productively infected human and abortively infected hamster cells. The specific recombination activity was increased in nuclear extracts of hamster cells abortively infected with Ad12. The junction sites in the recombinants, which were generated by the cell-free system, were very similar to junctions between adenoviral and cellular DNAs cloned from Ad12-induced tumor cells and Ad12-transformed cell lines.

Adenoviridae↗

PubMed1995

Alternative splicing generates a second isoform of the catalytic A subunit of the vacuolar H(+)-ATPase.

We have identified a second isoform of the catalytic A subunit of the vacuolar H+ pump in chicken osteoclasts. In this isoform (A2) a 72-bp cassette replaces a 90-bp cassette present in the classical A1 isoform. The A1-specific cassette encodes a region of the protein that contains one of the three ATP-binding consensus sequences (the P-loop) identified in this polypeptide, as well as the pharmacologically relevant Cys254. In contrast, the A2-specific cassette does not contain any of these features. These two isoforms, which appear to be ubiquitously expressed, are encoded by a single gene and are generated by alternative splicing of two mutually exclusive exons. The alternative RNA processing involves the recognition of a single site, the boundary between the A2- and A1-specific exons, as either acceptor (in A1) or donor (in A2) splice site.

Adenosine Triphosphate↗

PubMed1988

Cloning of the fusion gene of rinderpest virus: comparative sequence analysis with other morbilliviruses.

We have cloned the cDNA of the fusion (F) gene of the virulent (Kabete O) strain of rinderpest virus and provided a comparative analysis of its sequence with that of the F genes of measles and distemper viruses. The gene has an open reading frame of 2241 nucleotides with two potential initiation codons in-frame. Use of the first ATG would produce a polypeptide 747 amino acids long with a calculated molecular weight of 81,068. However, we suggest that the second ATG is used to generate the Fo protein, which is 546 amino acids long with a calculated molecular weight of 58,754. During maturation, the cleavage of F0 gives rise to the functional F1 and F2 polypeptides. The F1 polypeptide is 438 amino acids long and has a calculated molecular weight of 46,791, with a single (potential) glycosylation site in its cytoplasmic domain. The F2 polypeptide, probably 89 amino acids long after the signal sequence is cleaved, is estimated to be 9,800 Da and has three potential glycosylation sites. There is a divergence of 18.7% in amino acid sequences between rinderpest and measles virus F0 polypeptides; between distemper and rinderpest viruses the divergence is 31.8%. No significant homology in nucleotide sequences of rinderpest DNA to measles or distemper DNA was found in the 5' and 3' untranslated regions.

Base Sequence↗

PubMed2006

Discovery of a small-molecule HIV-1 integrase inhibitor-binding site.

Herein, we report the identification of a unique HIV-1 integrase (IN) inhibitor-binding site using photoaffinity labeling and mass spectrometric analysis. We chemically incorporated a photo-activatable benzophenone moiety into a series of coumarin-containing IN inhibitors. A representative of this series was covalently photo-crosslinked with the IN core domain and subjected to HPLC purification. Fractions were subsequently analyzed by using MALDI-MS and electrospray ionization (ESI)-MS to identify photo-crosslinked products. In this fashion, a single binding site for an inhibitor located within the tryptic peptide (128)AACWWAGIK(136) was identified. Site-directed mutagenesis followed by in vitro inhibition assays resulted in the identification of two specific amino acid residues, C130 and W132, in which substitutions resulted in a marked resistance to the IN inhibitors. Docking studies suggested a specific disruption in functional oligomeric IN complex formation. The combined approach of photo-affinity labeling/MS analysis with site-directed mutagenesis/molecular modeling is a powerful approach for elucidating inhibitor-binding sites of proteins at the atomic level. This approach is especially important for the study of proteins that are not amenable to traditional x-ray crystallography and NMR techniques. This type of structural information can help illuminate processes of inhibitor resistance and thereby facilitate the design of more potent second-generation inhibitors.

4-Hydroxycoumarins↗

PubMed1989

Primary structure and functional aspects of the gene coding for the second-largest subunit of RNA polymerase III of Drosophila.

We have cloned and sequenced the gene coding for the second-largest subunit of RNA polymerase III of Drosophila melanogaster (DmRP135). The gene, interrupted by two introns of 62 and 59 bp, respectively, codes for an mRNA of 3.6 kb. As for other housekeeping genes transcription initiates at several sites (between positions -98 and -76) none of which is preceded by a clear TATA sequence. The deduced polypeptide consists of 1129 amino acids with an aggregate molecular weight of 128 kDa. The protein sequence features the same regions of similarity as observed for the corresponding subunits of RNA polymerase II of Drosophila and yeast and the Escherichia coli beta subunit. As in the second-largest subunit of RNA polymerase II there is a zinc-binding motif which is absent in the beta subunit of E. coli. Antibodies directed against a fusion protein expressing 164 amino acids of the DmRP135 polypeptide cross-react with the second-largest subunit of RNA polymerase III of yeast and generate a distinct banding pattern on Drosophila polytene chromosomes distinguishable from that obtained with anti-RNA polymerase II antibodies.

Amino Acid Sequence↗

PubMed1994

Impact of premature stop codons on mRNA levels in infantile Sandhoff disease.

Sandhoff disease is an autosomal recessive lysosomal storage disease resulting from mutations of the HEXB gene encoding the beta subunit of beta-hexosaminidase A. Fibroblast lines from four patients with the infantile form of the disease were investigated for mutations by single strand conformation polymorphism analysis and direct sequencing of PCR products. Two of the cell lines were homozygous for a common, 16 kb deletion of the 5' end of HEXB gene. The two other cell lines contained the 16 kb deletion along with a second mutant allele generating a stop codon: in one case a nonsense mutation, C850-->T, which generated a stop codon at codon 284; and in the other, a single base deletion, delta T1344, which generated a stop codon at codon 451. One additional cell line investigated was a compound heterozygote for two frameshift mutations, delta G774 in exon 7 and delta AG1305-1306 in exon 11 (McInnes et al. 1992, Biochim. Biophys. Acta 1138: 315-317). Stop codons were generated in this cell line at codons 274 and 454, respectively. We took advantage of these genotypes to investigate the steady-state level of mRNA produced by cells containing stop codons using a competitive polymerase chain reaction technique. The mRNA levels were, as percent of normal per single gene dose: for the stop codon at codon 451, 30%; for those at codons 274 and 454, combined percentage of 1.7%; and at codon 284, 0.8%. These studies demonstrate a dramatic difference in the steady-state level of Hex beta mRNA in the cell lines with stop codons in close proximity to each other (codons 451 vs 454).(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1998

Proprioceptive control of posture: a review of new concepts.

The assumption that proprioceptive inputs from the lower legs are used to trigger balance and gait movements is questioned in this review (an outgrowth of discussions initiated during the Neural Control of Movement Satellite meeting held in Cozumel, Mexico, April 1997). Recent findings presented here suggest that trunk or hip inputs may be more important in triggering human balance corrections and that proprioceptive input from the lower legs mainly helps with the final shaping and intermuscular coordination of postural and gait movements. Three major questions were considered. First, what role, if any, do lower-leg proprioceptive inputs play in the triggering of normal balance corrections? If this role is negligible, which alternative proprioceptive inputs then trigger balance corrections? Second, what is the effect of proprioceptive loss on the triggering of postural and gait movements? Third, how does proprioceptive loss affect the output of central pattern generators in providing the final shaping of postural movements? The authors conclude that postural and gait movements are centrally organized at two levels. The first level involves the generation of the basic directional-specific response pattern based primarily on hip or trunk proprioceptive input secondarily on vestibular inputs. This pattern specifies the spatial characteristics of muscle activation, that is which muscles are primarily activated, as well as intermuscular timing, that is, the sequence in which muscles are activated. The second level is involved in the shaping of centrally set activation patterns on the basis of multisensorial afferent input (including proprioceptive input from all body segments and vestibular sensors) in order that movements can adapt to different task conditions. Copyright 1998 Elsevier Science B.V.

Journal Article↗

PubMed1994

Analysis of RNA chain elongation and termination by Saccharomyces cerevisiae RNA polymerase III.

When Saccharomyces cerevisiae RNA polymerase (Pol) III transcribes the S. cerevisiae SUP4 tRNA(Tyr) gene, it is obliged to navigate past a large, multi-subunit DNA-bound complex of proteins. We have analyzed individual steps of RNA chain elongation on this gene. Slow steps of transcriptional initiation were by-passed by forming 5'-end-labeled, arrested and precisely positioned transcription complexes. Synchronous resumption of chain elongation by these complexes allowed a single round of RNA synthesis and termination to be analyzed in detail. Results for synthesis at 20 degrees C and 0 degrees C, in the presence of 100 microM and 1 mM ribonucleoside triphosphates (NTPs) are presented. RNA chain elongation through assembled transcription complexes was uneven but relatively rapid: at 20 degrees C with 1 mM NTPs, the fastest RNA chains elongated at an average rate of 29 nucleotides (nt)/second, and the median RNA chains elongated at 21 to 22 nt/second on average. These rates are comparable with a recent measurement of the average rate of chain elongation in vivo by Drosophila RNA polymerase II at 25 degrees C. At 0 degree C, RNA chain elongation rates were, on average, approximately 30-fold slower. Quantitative analysis of the individual steps of RNA chain elongation showed that steps of adding U and A to U-terminated RNA chains tended to be relatively slow, and to be more strongly influenced by nucleotide concentration. Termination of transcription occurred in the sequence T7GT6 (in the non-template DNA strand) and was progressive. Transcripts with five, six and seven U residues were formed, and there was even slow readthrough of the T7 stretch, with GU3 adding rapidly, suggesting that incorporation of a single G into the RNA chain served to reset elongation rates substantially or entirely. Stripping transcription factor (TF) IIIC from transcription complexes did not substantially increase overall RNA chain growth rate, but did diminish pausing at a single site upstream of the boxB binding site of TFIIIC. The TFIIIC-generated delay at this single site was estimated to be only approximately 0.15 to 0.2 seconds at 20 degrees C. Quantitative analysis of RNA chain elongation yielded kinetic parameters for the individual steps of nucleotide addition that were used in computer simulations of RNA chain growth. Elongation modeled as a simple sequence of pseudo-first-order reactions yielded computed RNA chain length distributions that remained relatively synchronous during elongation, while observed chain growth quickly became desynchronized.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1999

Molecular cloning and sequence analysis of the phosphoprotein (P) gene of the lapinized rinderpest virus.

We determined the nucleotide sequence of the coding region for the phosphoprotein (P) gene of the L strain of rinderpest virus (RPV). The gene encodes two overlapping open reading frames of 1521 and 531 nucleotides. Use of the first ATG would produce a P polypeptide of 507 amino acids, while use of the second ATG would produce a C polypeptide of 177 amino acids. In addition, the insertion of an extra G residue at the editing site generates an alternative mRNA potentially encoding the V protein of RPV. Homology comparisons of the P, C and V proteins among various viruses suggest that RPV is closer to measles virus (MV) than to canine distemper virus (CDV). Alignment of the sequences unique to the V protein revealed that the cysteine residues are well conserved among RPV, MV and CDV, and form a "zinc finger"-like motif.

Amino Acid Sequence↗

PubMed1998

Sequence-dependent antitumour efficacy of combination chemotherapy of nedaplatin, a novel platinum complex, with 5-fluorouracil in an in vivo murine tumour model.

The antitumour efficacy of a sequential combination of nedaplatin (NDP) and 5-fluorouracil (5-FU) was evaluated using Lewis lung carcinoma in vivo. NDP was developed as a second generation platinum complex. Because it has greater antitumour activity and lower nephrotoxicity than cisplatin (CDDP), we also compared the antitumour activity of NDP plus 5-FU with that of CDDP plus 5-FU. A fixed 5-FU dose was injected daily for 5 days and increasing doses of either NDP or CDDP were injected once via the tail vein into the Lewis lung carcinoma-implanted mice. The sequential administration of either NDP or CDDP prior to 5-FU (NF or CF therapy) resulted in severe body weight loss followed by the death of the tumour-bearing mice when the high-dose of NDP or CDDP was administered. In contrast, the sequential administration of 5-FU prior to NDP or CDDP (FN or FC therapy) resulted in synergistically enhanced inhibition of tumour growth and prolonged survival in comparison with NDP, CDDP or 5-FU monotherapy. At the high-dose of NDP in FN therapy, a reduction of tumour size and long-term tumour-free survival were frequently observed. The survival effect of the combinations of NDP with 5-FU was superior to those of the combination of CDDP with 5-FU. In conclusion, the sequence-dependent antitumour efficacy and toxicity of the combination of NDP or CDDP with 5-FU was demonstrated in this study, and FN therapy appeared to be the most efficient regimen as a clinical therapy.

Animals↗

PubMed2005

An outbreak of SHV-5 producing Klebsiella pneumoniae in a neonatal intensive care unit; meropenem failed to avoid fecal colonization.

An outbreak of extended-spectrum beta-lactamase (ESBL) producing Klebsiella pneumoniae (ESBL-Kp) in a neonatal intensive care unit prompted a prospective surveillance study between 12th September and 6th October 2003. Surveillance was carried out by obtaining stool samples twice a week. The DNA relatedness of the isolates was shown by random amplified polymorphic DNA comparison (ERIC-PCR). ESBL production was identified by clavulanate synergy, isoelectric focusing, PCR and sequence analysis. During the study period, 49 neonates were hospitalized in the neonatal intensive care unit (NICU). In the first 20-day period, five neonates were infected with ESBL-Kp. The first patient treated with third generation cephalosporin and the second patient treated with meropenem died. While all three infected survivors were clinically improving, the digestive tracts were being colonized by SHV-5 producing Klebsiella. In the next period of the study, five neonates were colonized by ESBL-Kp as well. Univariate comparison of risk factors between colonized and non-colonized neonates was not significant. A total of 24 colonally related ESBL-Kp have been recovered from clinical materials and stool samples. This study demonstrated that parenterally applied meropenem, though successful in treating the systemic illness, might fail to protect the digestive tract from colonization of ESBL-Kp.

Anti-Bacterial Agents↗

PubMed1987

Isolation of anonymous DNA sequences from within a submicroscopic X chromosomal deletion in a patient with choroideremia, deafness, and mental retardation.

Choroideremia, an X-chromosome linked retinal dystrophy of unknown pathogenesis, causes progressive nightblindness and eventual central blindness in affected males by the third to fourth decade of life. Choroideremia has been mapped to Xq13-21 by tight linkage to restriction fragment length polymorphism loci. We have recently identified two families in which choroideremia is inherited with mental retardation and deafness. In family XL-62, an interstitial deletion in Xq21 is visible by cytogenetic analysis and two linked anonymous DNA markers, DXYS1 and DXS72, are deleted. In the second family, XL-45, an interstitial deletion was suspected on phenotypic grounds but could not be confirmed by high-resolution cytogenetic analysis. We used phenol-enhanced reassociation of 48,XXXX DNA in competition with excess XL-45 DNA to generate a library of cloned DNA enriched for sequences that might be deleted in XL-45. Two of the first 83 sequences characterized from the library were found to be deleted in probands from family XL-45 as well as from family XL-62. Isolation of these sequences proves that XL-45 does contain a submicroscopic deletion and provides a starting point for identifying overlapping genomic sequences that span the XL-45 deletion. Each overlapping sequence will be studied to identify exons from the choroideremia locus.

Chromosome Mapping↗

PubMed1998

Menin mutations in the diagnosis and prediction of multiple endocrine neoplasia type 1.

INTRODUCTION: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the development of multiple endocrine adenomas, typically in the pancreas, anterior pituitary, and parathyroid glands. The disease is associated with germ-line mutations of the menin gene, a putative tumor-suppressor gene located on human chromosome 11q13. METHODS: To facilitate the diagnosis and prediction of MEN1 in patients and their relatives, we developed a molecular two-step strategy to screen for menin gene mutations. DNA fragments covering the entire menin coding sequence are generated from patient cDNA by polymerase reaction (PCR) and subsequently analyzed by single-strand conformational polymorphism electrophoresis (SSCP). Fragments with aberrant SSCP migration are DNA-sequenced to directly characterize menin mutations. In a second diagnostic step, genomic DNA of healthy relatives of the corresponding MEN1 index patient is analyzed by PCR, with only the specific exon amplified harboring the family-specific mutation. Mutation-specific restriction enzyme digestion of this PCR product finally allows the identification of mutation carriers through pathological restriction fragment patterns. RESULTS: Using this approach, we identified an in-frame deletion mutation (delta Tyr Met) located in menin exon 4 (codon 227-228) that co-segregates with the disease phenotype in a large MEN1 family from Southern Germany. CONCLUSION: It is likely that the direct molecular analysis of menin gene mutations will replace the genetic and biochemical screening tests currently used in the clinical management of MEN1 families. In addition, these studies may provide clues to the tumor biology of both sporadic and MEN1-associated endocrine adenomas.

Adult↗

PubMed2004

Sequential activation of microcircuits underlying somatosensory-evoked potentials in rat neocortex.

Evoked cortical field potentials are widely used in neurophysiological studies into cortical functioning, but insight in the underlying neural mechanisms is severely hampered by ambiguities in the interpretation of the field potentials. The present study aimed at identifying the precise relationships between the primary evoked cortical field potential (the positive-negative [P1-N1]response) and the temporal and spatial sequence in which different local cortical micro-circuits are recruited. We electrically stimulated the median nerve and recorded field potentials using a 12-channel depth probe in somatosensory cortex of ketamine anesthetized rats. Current source density analysis was used and a grand average was constructed based on all individual animals taking into account individual differences in cortical layering. Manipulation of stimulus strength, selective averaging of single trial responses, and double-pulse stimulation, were used to help disentangle overlapping dipoles and to determine the sequence of neuronal events. We discriminated three phases in the generation of the P1-N1 wave. In the first phase, specific thalamic afferents depolarize both layer III and layer V pyramidal cells. In the second phase, superficial pyramidal cells are depolarized via supragranular intracortical projections. In the third phase, population spikes are generated in layer Vb pyramidal cells, associated with a distinct fast (approximately 1 ms) sink/source configuration. Axon-collaterals of layer Vb pyramidal cells produce an enhanced activation of the supragranular pyramidal cells in layer I-II, which generates N1.

Algorithms↗

PubMed1999

Mutational analysis of the RET proto-oncogene in a kindred with multiple endocrine neoplasia type 2A and Hirschsprung's disease.

BACKGROUND/PURPOSE: Germline mutations of the RET proto-oncogene (RET; 10q11.2) have been reported in multiple endocrine neoplasia type 2A (MEN 2A) and Hirschsprung's disease. The authors investigated a Japanese kindred in which MEN 2A and Hirschsprung's disease frequently have been found. METHODS: The pedigree consisted of 28 members (11 boys and 17 girls) spanning 4 generations, of whom, 8 were affected with MEN 2A or Hirschsprung's disease. RESULTS: Direct sequence DNA analysis of the RET proto-oncogene showed a heterozygosity for a G to C transition at the second nucleotide of codon 620 (exon 10) in the patients, resulting in the replacement of cysteine by a serine residue in the affected Ret protein. This family added a novel RET missense mutation (C620S) predisposing to the association of MEN 2A and Hirschsprung's disease. CONCLUSION: Detection of the mutated RET gene carriers may be used for genetic counseling of potential risk for Hirschsprung's disease as well as MEN 2A in the affected families.

Codon↗

PubMed2001

The structure of a pyoverdine produced by a Pseudomonas tolaasii-like isolate.

Cultures of Agaricus bisporus, the most extensively cultivated mushroom, can be infected severely by Pseudomonas tolaasii. This pathogen is characterized by the so-called white line reaction, a precipitate formed on agar plates between its colonies and those of P. reactans, both belonging to the collective species P. fluorescens. A recent study has shown that a group of P. tolaasii isolates can be subdivided into two groups or 'siderovars', based on the pyoverdines they produce (Munsch et al. 2000). One group of strains is characterized by the pyoverdine described by Demange et al. (1990). A representative of the second group (strain Ps3a) was found to produce the same pyoverdine as a strain which had been classified before as P. aureofaciens. However, based mainly on 16S rRNA gene sequence comparisons and REP-PCR generated fingerprints, the two strains are not identical. They are also distinguishable from the P. tolaasii type strain.

Agaricales↗

PubMed2004

Intron 4 mutation in APC gene results in splice defect and attenuated FAP phenotype.

The adenomatous polyposis coli (APC) protein is a tumor suppressor frequently involved in the development of inherited and sporadic colon cancers. Somatic mutations of the APC gene are found in 80% of all colon cancers. Inherited mutations result in familial adenomatous polyposis (FAP) as well as an attenuated form of this syndrome. FAP is characterized by the early age onset of hundreds to thousands of colonic adenomatous polyps and a virtual certainty of colon cancer unless the colon is removed. The attenuated form of FAP (AFAP) is characterized by fewer adenomas, later onset of adenomas and cancer, and a decreased lifetime cancer risk. We report a 37-year-old man with a history of more than 50 colonic adenomatous polyps, located predominately in the right colon. An insertion of a single thymidine between the second and third base pairs of intron 4 of the APC gene was identified (c.531+2_531+3insT). Monoallelic hybrid cells harboring a single copy of human chromosome 5 were generated from patient lymphoblasts. Sequencing of the APC cDNA product from these cells revealed a single RNA transcript with aberrant splicing in the mutant mRNA whereby exon 4 is deleted. The translational reading frame is shifted after codon 140 and a translational stop is generated predicting a truncated protein of 147 amino acids, thus indicating that the intronic mutation is disease causing. The lack of a secondary transcript from the mutant allele suggests that incomplete exon skipping is not the molecular mechanism behind the attenuated phenotype.

Adenomatous Polyposis Coli↗