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PubMed2005

[Modification of hybrid antimicrobial peptide CecA-mil gene and its over-secretion expression in Pichia pastoris].

According to the partiality codon of Pichia pastoris, hybrid antimicrobial peptide CecA-mil gene was reconstructed, synthesized and cloned into pPICZalpha-A to construct the recombinant expression vector pPICZa-A-CM. The pPICZalpha-A-CM was transformed into yeast host strain X-33. Under the control of the promoter AOX1 (alcohol oxidase 1), a approximately 1.9 kD cecA-mil protein was expressed with the high level of 245 microg/mL after optimized the requirements for the flask-shaking culture fermentation of the Pichia pastoris rX-33/pPICZalpha-A-CM. The hybrid antibacterial peptide had a broad spectrum antibacterial activity on both gram-positive and gram-negative bacteria, especially showed potent antibacterial activity against ampicillin-resistant and kanamycin- resistant bacteria, such as Staphylococcus aureus (cowan I) and pathogenic E. coli (O1) from chicken. In addition, the hybrid antibacterial peptide showed an extreme heat-stable and acid-stable characteristic. Based on these characteristics, the recombinant antibacterial peptide CecA-mil display application foreground in the field of antisepsis of food, prevention of disease, additives of animal feedstuff and so on.

Anti-Infective Agents↗
PubMedDate not supplied

Compositional patterns in vertebrate genomes: conservation and change in evolution.

The evolution of vertebrate genomes can be investigated by analyzing their regional compositional patterns, namely the compositional distributions of large DNA fragments (in the 30-100-kb size range), of coding sequences, and of their different codon positions. This approach has shown the existence of two evolutionary modes. In the conservative mode, compositional patterns are maintained over long times (many million years), in spite of the accumulation of enormous numbers of base substitutions. In the transitional, or shifting, mode, compositional patterns change into new ones over much shorter times. The conservation of compositional patterns, which has been investigated in mammalian genomes, appears to be due in part to some measure of compositional conservation in the base substitution process, and in part to negative selection acting at regional (isochore) levels in the genome and eliminating deviations from a narrow range of values, presumably corresponding to optimal functional properties. On the other hand, shifts of compositional patterns, such as those that occurred between cold-blooded and warm-blooded vertebrates, appear to be due essentially to both negative and positive selection again operating at the isochore level, largely under the influence of changes in environmental conditions, and possibly taking advantage of mutational biases in the replication/repair enzymes and/or in the enzyme make-up of nucleotide precursor pools. Other events (like translocations and changes in chromosomal structure) also play a role in the transitional mode of genome evolution. The present findings (1) indicate that isochores, which correspond to the DNA segments of individual or contiguous chromatin domains, represent selection units in the vertebrate genome; and (2) shed new light on the selectionist-neutralist controversy.

Animals↗
PubMed2002

No accident: genetic codes freeze in error-correcting patterns of the standard genetic code.

The standard genetic code poses a challenge in understanding the evolution of information processing at a fundamental level of biological organization. Genetic codes are generally coadapted with, or 'frozen' by, the protein-coding genes that they translate, and so cannot easily change by natural selection. Yet the standard code has a significantly non-random pattern that corrects common errors in the transmission of information in protein-coding genes. Because of the freezing effect and for other reasons, this pattern has been proposed not to be due to selection but rather to be incidental to other evolutionary forces or even entirely accidental. We present results from a deterministic population genetic model of code-message coevolution. We explicitly represent the freezing effect of genes on genetic codes and the perturbative effect of changes in genetic codes on genes. We incorporate characteristic patterns of mutation and translational error, namely, transition bias and positional asymmetry, respectively. Repeated selection over small successive changes produces genetic codes that are substantially, but not optimally, error correcting. In particular, our model reproduces the error-correcting patterns of the standard genetic code. Aspects of our model and results may be applicable to the general problem of adaptation to error in other natural information-processing systems.

Animals↗
PubMed1993

The Escherichia coli K-12 "wild types" W3110 and MG1655 have an rph frameshift mutation that leads to pyrimidine starvation due to low pyrE expression levels.

The widely used and closely related Escherichia coli "wild types" W3110 and MG1655, as well as their common ancestor W1485, starve for pyrimidine in minimal medium because of a suboptimal content of orotate phosphoribosyltransferase, which is encoded by the pyrE gene. This conclusion was based on the findings that (i) the strains grew 10 to 15% more slowly in pyrimidine-free medium than in medium containing uracil; (ii) their levels of aspartate transcarbamylase were highly derepressed, as is characteristic for pyrimidine starvation conditions; and (iii) their levels of orotate phosphoribosyltransferase were low. After introduction of a plasmid carrying the rph-pyrE operon from strain HfrH, the growth rates were no longer stimulated by uracil and the levels of aspartate transcarbamylase were low and similar to the levels observed for other strains of E. coli K-12, E. coli B, and Salmonella typhimurium. To identify the mutation responsible for these phenotypes, the rph-pyrE operon of W3110 was cloned in pBR322 from Kohara bacteriophage lambda 2A6. DNA sequencing revealed that a GC base pair was missing near the end of the rph gene of W3110. This one-base-pair deletion results in a frame shift of translation over the last 15 codons and reduces the size of the rph gene product by 10 amino acid residues relative to the size of RNase PH of other E. coli strains, as confirmed by analysis of protein synthesis in minicells. The truncated protein lacks RNase PH activity, and the premature translation stop in the rph cistron explains the low levels of orotate phosphoribosyltransferase in W3110, since close coupling between transcription and translation is needed to support optimal levels of transcription past the intercistronic pyrE attenuator.

Amino Acid Sequence↗
PubMed1993

[Aromatase activities of endometrial carcinomas and both basic and clinical analyses of endometrial hyperplasia as a premalignant disease].

Paraffin-embedded materials obtained from 117 cases of endometrial hyperplasia and 84 cases of carcinoma were used for measurement of both ki-ras and p53 gene mutation and aromatase (ARO) and TGF-alpha immunostaining. The overall incidence of ki-ras mutations in the hyperplasia specimens (16%) was similar to the incidence detected in carcinomas (18%). None of 117 endometrial hyperplasias were found to have mutations in the p53 gene, whereas mutations were seen in 3 (13.3%) endometrial carcinomas. The intensity of both ARO and TGF-alpha immunostaining was increased in glands of both hyperplasia and carcinoma, and also in the interstitium of carcinoma. The positive sites of both ARO and TGF-alpha were almost the same, with an incidence below 40% in both hyperplasias and carcinomas. The cultured cells of endometrial carcinoma showed aromatase activity below MCF-7 cells, because testosterone was converted to estradiol (E2). TGF-alpha induced cell growth with at an optimal concentration. In HEC-59 cells, TGF-alpha increased both ARO-activity and mRNA. Some promoters on ARO-exon 1 in HEC-59 cells were different from those in BeWo cells. Progesterone inhibited the E2-induced excretion of pre TGF-alpha in endometrial carcinoma cells. These findings suggest that endometrial hyperplasia can be a premalignant condition of carcinoma, and can be initiated by both ki-ras codon 12 mutation and abnormal activity of ARO induced by TGF-alpha. In addition, HEC-59 cells may possess autocrine/paracrine properties involving ARO, E2 and TGF-alpha.

Adult↗
PubMed2004

Mycoplasma hyopneumoniae p65 surface lipoprotein is a lipolytic enzyme with a preference for shorter-chain fatty acids.

Mycoplasma hyopneumoniae is the most significant bacterial pathogen of the respiratory tract of swine. p65 is an immunodominant surface lipoprotein of M. hyopneumoniae that is specifically recognized during disease. Analysis of the translated amino acid sequence of the gene encoding p65 revealed similarity to the GDSL family of lipolytic enzymes. To examine the lipolytic activity of p65, the gene was cloned and expressed in Escherichia coli after truncation of the prokaryotic lipoprotein signal sequence and mutagenesis of the mycoplasma TGA tryptophan codons. After treatment with thrombin, the recombinant glutathione S-transferase (GST)-p65 protein yielded a 66-kDa fusion protein cleavage product corresponding in size to the mature p65 protein. The esterase activity of recombinant GST-p65 was indicated by the formation of a cleared zone on tributyrin agar plates and the hydrolysis of p-nitrophenyl esters of caproate (pNPC) and p-nitrophenyl esters of palmitate (pNPP). Lipase activity was indicated by the hydrolysis of the artificial triglyceride 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester. Using pNPC and pNPP as substrates, recombinant GST-p65 had optimal activity between pHs 9.2 and 10.2 and at a temperature higher than 39 degrees C. Calcium ions did not increase the activity of recombinant GST-p65. Rabbit anti-p65 antibodies inhibited the activity of recombinant GST-p65 and also inhibited the growth of M. hyopneumoniae in vitro. Examination of the kinetic parameters of recombinant GST-p65 for the hydrolysis of pNPC and pNPP indicated a preference for the shorter fatty acid chain of pNPC. The physiological and/or pathogenic role of mycoplasma lipolytic enzymes has not been determined, but they are likely to play an important role in mycoplasmas' nutritional requirements for long-chain fatty acids and may reduce the function of lung surfactants in mycoplasma-induced respiratory diseases. This is the first report of the lipolytic activity of a lipid-modified surface immunogen of a mycoplasma.

Amino Acid Sequence↗
PubMed2005

Rapid and easy thermodynamic optimization of the 5'-end of mRNA dramatically increases the level of wild type protein expression in Escherichia coli.

Low levels of expression in Escherichia coli are often observed when using wild type proteins. The addition of an N-terminal His-tag to these same proteins dramatically improves the level of expression. We therefore concluded that post-transcriptional regulation and in particular translational regulation are probably influenced by the presence of the tag. The RNAfold program was used to analyze the 5'-end of the encoding mRNA, and more precisely the area encompassing the Shine-Dalgarno region and the initiation codon ATG. We observed that hairpin loops can be formed and that the stability of these loops correlates with the level of protein expression in E. coli. Our recently developed cloning technology by PCR fragment integration allows us to easily and rapidly introduce mutations anywhere within a gene. In our studies, we used this technology to destabilize the predicted hairpin by introducing silent mutations within the first 72 nucleotides of the coding sequence. As a result of the decreased stability of the RNA hairpins, we could significantly increase the level of expression of wild type proteins and without having to rely on the use of tags in E. coli. In addition, our studies allow us to predict whether or not a protein will be expressed without additional engineering of its encoding gene.

5' Untranslated Regions↗
PubMed2003

[Gene optimization is necessary to express HPV type 6 L1 protein in the methylotrophic yeast Pichia pastoris].

OBJECTIVE: Human papillomavirus 6 (HPV 6) causes genital warts, a common sexually transmitted disease. L1-capsids protein is a highly promising vaccine candidate that has entered phase II clinical trial. But the existing methodologies for producing L1-capsids in insect cells is expensive for use in developing countries. As methylotrophic yeast,the Pichia pastoris expression system offers economy,and high expression levels. Over-expression of HPV6-L1 protein in P. pastoris was the purpose of this study. METHODS: The whole L1 gene with preferred codons for P. pastoris was rebuilt and A-T rich regions were abolished, Cloning into pPIC3.5K,electroporation of KM71, in vivo screen of multiple inserts by G418 resistance, PCR analysis of pichia integrants, BMGY/BMMY are used for induction and expression of L1 proteins. RESULTS: Three clones were found to produce L1 protein which can be identified with Western blot. Compared with L1 protein from E.coli, pichia-produced L1 has some glycosylation. Reacting strongly with MabH6B10.5 in indirect immunofluorescence assay indicated that L1 protein expressed in pichia cell holds its native conformational epitopes which is important for vaccine use. A total 125 microg pure L1 protein could be obtained from 1L cultures through ion-exchange and Ni-NTA chromatography. CONCLUSION: HPV type 6 L1 protein expressed in Pichia pastoris will facilitate the HPV vaccine development and structure-function study.

Capsid Proteins↗
PubMed1985

The promoter of the late p10 gene in the insect nuclear polyhedrosis virus Autographa californica: activation by viral gene products and sensitivity to DNA methylation.

In lepidopteran insect cells infected with the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), two major late viral gene products are expressed: the polyhedrin, a 28 000 mol. wt. protein which makes up the mass of the nuclear inclusion bodies, and a 10 000 mol. wt. protein (p10) whose function is unknown. The nucleotide sequences of these strong promoters conform to those of other eukaryotic promoters and are rich in AT base pairs. We used the pSVO-CAT construct containing the prokaryotic gene chloramphenicol acetyl transferase (CAT) to study the function of the p10 gene promoter in insect and mammalian cells. Upon transfection of the pAcp10-CAT construct, which contained 402 bp of the p10 gene of AcNPV DNA in the HindIII site of pSVO-CAT, CAT activity was determined. The p10 gene promoter was inactive in human HeLa cells and in uninfected Spodoptera frugiperda insect cells. The same promoter was active, however, in AcNPV-infected S. frugiperda cells and exhibited optimal activity when cells were transfected 18 h after infection with the insect virus. This finding demonstrated directly that the p10 gene promoter required other viral gene products for its activity in insect cells. The nature of these products was unknown. The p10 gene promoter sequence contained one 5'-CCGG-3' site 40 bp upstream from the cap site of the gene and two such sites 178 and 192 bp downstream from the ATG initiation codon of the gene. Since Drosophila DNA or S. frugiperda DNA contained no 5-methylcytosine or extremely small amounts of it, we were interested in determining the effect of site-specific methylations on the p10 gene insect virus promoter. Methylation at the 5'-CCGG-3' sites led to a block of this promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyltransferases↗
PubMed2005

Evaluation of an oligonucleotide ligation assay for detection of mutations in HIV-1 subtype C individuals who have high level resistance to nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors.

The oligonucleotide ligation assay (OLA) has been proposed as an affordable alternative to sequence-based HIV-1 drug resistance testing in resource poor settings. The aim was to evaluate OLA for detecting mutations K103N, Y181C, K65R, Q151M, M184V and T215Y/F in subtype C. Forty-four subtype C and 8 subtype B HIV-1 positive individuals were analysed using the ViroSeqtrade mark HIV-1 genotyping assay (Applied Biosystems, Foster City, CA). A one-step RT-PCR and nested PCR were performed using subtype B specific primers from the OLA kit (NIH AIDS Research and Reference Reagent Program). Seventy-eight subtype C sequences were used to design subtype C specific primers. Ligation and detection steps were followed according to OLA kit protocol. For codons, K103N, Y181C, K65R, Q151M, M184V and T215Y/F, four or more mismatches compared to the probe or mismatches less than four bases from the ligation site were not tolerated. Results revealed accurate identification of mutations in 2/10, 4/9 3/9, 6/7, 2/7 and 6/7 VQA samples and 5/20, 4/17 0/20, 18/24, 5/24 and 13/24 subtype C positive individuals, respectively. It was concluded that the probes and primers in the NIH reference kit would need modification to optimize detection of mutations in subtype C individuals.

Anti-HIV Agents↗
PubMed2006

Molecular basis of Kindler syndrome in Italy: novel and recurrent Alu/Alu recombination, splice site, nonsense, and frameshift mutations in the KIND1 gene.

Kindler syndrome (KS) is a rare autosomal recessive disorder characterized by skin blistering in childhood followed by photosensitivity and progressive poikiloderma. Most cases of KS result from mutations in the KIND1 gene encoding kindlin-1, a component of focal adhesions in keratinocytes. Here, we report novel and recurrent KIND1 gene mutations in nine unrelated Italian KS individuals. A novel genomic deletion of approximately 3.9 kb was identified in four patients originating from the same Italian region. This mutation deletes exons 10 and 11 from the KIND1 mRNA leading to a truncated kindlin-1. The deletion breakpoint was embedded in AluSx repeats, specifically in identical 30-bp sequences, suggesting Alu-mediated homologous recombination as the pathogenic mechanism. KIND1 haplotype analysis demonstrated that patients with this large deletion were ancestrally related. Five additional mutations were disclosed, two of which were novel. To date, four recurrent mutations have been identified in Italian patients accounting for approximately approximately 75% of KS alleles in this population. The abundance of repetitive elements in intronic regions of KIND1, together with the identification of a large deletion, suggests that genomic rearrangements could be responsible for a significant proportion of KS cases. This finding has implications for optimal KIND1 mutational screening in KS individuals.

Adolescent↗
PubMed2004

Accuracy and power of statistical methods for detecting adaptive evolution in protein coding sequences and for identifying positively selected sites.

The parsimony method of Suzuki and Gojobori (1999) and the maximum likelihood method developed from the work of Nielsen and Yang (1998) are two widely used methods for detecting positive selection in homologous protein coding sequences. Both methods consider an excess of nonsynonymous (replacement) substitutions as evidence for positive selection. Previously published simulation studies comparing the performance of the two methods show contradictory results. Here we conduct a more thorough simulation study to cover and extend the parameter space used in previous studies. We also reanalyzed an HLA data set that was previously proposed to cause problems when analyzed using the maximum likelihood method. Our new simulations and a reanalysis of the HLA data demonstrate that the maximum likelihood method has good power and accuracy in detecting positive selection over a wide range of parameter values. Previous studies reporting poor performance of the method appear to be due to numerical problems in the optimization algorithms and did not reflect the true performance of the method. The parsimony method has a very low rate of false positives but very little power for detecting positive selection or identifying positively selected sites.

Algorithms↗
PubMed1997

Catalytic properties and sensitivity to tentoxin of Chlamydomonas reinhardtii ATP synthases changed in codon 83 of atpB by site-directed mutagenesis.

The participation of the amino acid beta83 in determining the sensitivity of chloroplast ATP synthases to tentoxin was reported previously. We have changed codon 83 of the Chlamydomonas reinhardtii atpB gene by site-directed mutagenesis to further examine the role of this amino acid in the response of the ATP synthase to tentoxin and in the mechanism of ATP synthesis and hydrolysis. Amino acid beta83 was changed from Glu to Asp (betaE83D) and to Lys (betaE83K), and the highly conserved tetrapeptide betaT82-E83-G84-L85 (DeltaTEGL) was deleted. Mutant strains were produced by particle gun transformation of atpB deletion mutants cw15DeltaatpB and FUD50 with the mutated atpB genes. The transformants containing the betaE83D and betaE83K mutant genes grew well photoautotrophically. The DeltaTEGL transformant did not grow photoautotrophically, and no CF1 subunits were detected by immunostaining of Western blots using CF1 specific antibodies. The rates of ATP synthesis at clamped DeltapH with thylakoids isolated from cw15 and the two mutants, betaE83D and betaE83K, were similar. However, only the phosphorylation activity of the mutant betaE83D was inhibited by tentoxin with 50% inhibition attained at 4 microM. These results confirm that amino acid beta83 is critical in determining the response of ATP synthase to tentoxin. The rates of the latent Mg-ATPase activity of the CF1s isolated from cw15, betaE83D, and betaE83K were similar and could be enhanced by heat, alcohols, and octylglucoside. As in the case of the membrane-bound enzyme, only CF1 from the betaE83D mutant was sensitive to tentoxin. A lower alcohol concentration was required for optimal stimulation of the ATPase of the betaE83K-CF1 than that of CF1 from the other two strains. Moreover, the optimal activity of the betaE83K-CF1 was also lower. These results suggest that introduction of an amino acid with a positively charged side chain in position 83 in the "crown" domain affects the active conformation of the CF1-ATPase.

ATP Synthetase Complexes↗
PubMed2003

Cloning, expression, and purification of His-tagged rat mevalonate kinase.

Mevalonate kinase catalyzes the phosphorylation of mevalonic acid to form mevalonate 5-phosphate, which plays a key role in regulating cholesterol biosynthesis in animal cells. Deficiency of mevalonate kinase activity in the human body has been linked to mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS). We cloned the gene of rat mevalonate kinase into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5(') of the gene. The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel HiTrap chelating metal affinity column in 90% yield to apparent homogeneity. The purified rat mevalonate kinase had a dimeric structure composed of identical subunits. Based on SDS-PAGE, the subunit was 42 kDa. The specific activity of the purified His-tagged rat mevalonate kinase was 32.7 micromol/min/mg and the optimal pH was found to be 7.0-8.0 in phosphate buffer. The Michaelis constant K(M) was 35 microM for (RS)-mevalonate and 953 microM for ATP, respectively. The V(max) was determined to be 38.7 micromol/min/mg. The overexpression of rat mevalonate kinase in E. coli and one-step purification of the highly active rat mevalonate kinase will facilitate further our investigation of this enzyme through site-directed mutagenesis and enzyme-catalyzed reactions with substrate analogs.

Animals↗
PubMed2003

Two different mechanisms for ribosome/mRNA interaction in archaeal translation initiation.

In this study, we have analysed the features of mRNA/ribosome interaction in the thermophilic archeon Sulfolobus solfataricus. Leadered mRNAs endowed with ShineDalgarno (SD) motifs formed stable binary complexes with 30S subunits, optimally at high temperature (6570 degrees C) and without the aid of initiator tRNA (tRNAi) or any factor. 'Toeprinting' assays revealed that the SD motifs were necessary and sufficient to direct the 30S subunit to the translation initiation region. Leaderless mRNAs, i.e. mRNAs entirely lacking a 5'-untranslated region (UTR), did not interact directly with 30S subunits but required the presence of tRNAi, indicating that codonanticodon pairing was required for positioning the ribosome on the initiation codon. The data suggest that archaea such as Sulfolobus routinely use two distinct mechanisms for translational initiation. SD-dependent initiation, resembling the pathway prevalent in present-day bacteria, would operate on distal cistrons of polycistronic mRNAs, whereas 'leaderless' initiation, reminiscent of the eukaryotic pathway, would operate on monocistronic mRNAs and on opening cistrons of polycistronic mRNAs.

5' Untranslated Regions↗
PubMed1994

Purification and characterization of Streptococcus sobrinus dextranase produced in recombinant Escherichia coli and sequence analysis of the dextranase gene.

The plasmid (pYA902) with the dextranase (dex) gene of Streptococcus sobrinus UAB66 (serotype g) produces a C-terminal truncated dextranase enzyme (Dex) with a multicomplex mass form which ranges from 80 to 130 kDa. The Escherichia coli-produced enzyme was purified and characterized, and antibodies were raised in rabbits. Purified dextranase has a native-form molecular mass of 160 to 260 kDa and specific activity of 4,000 U/mg of protein. Potential immunological cross-reactivity between dextranase and the SpaA protein specified by various recombinant clones was studied by using various antisera and Western blot (immunoblot) analysis. No cross-reactivity was observed. Optimal pH (5.3) and temperature (39 degrees C) and the isoelectric points (3.56, 3.6, and 3.7) were determined and found to be similar to those for dextranase purified from S. sobrinus. The dex DNA restriction map was determined, and several subclones were obtained. The nucleotide sequence of the dex gene was determined by using subclones pYA993 and pYA3009 and UAB66 chromosomal DNA. The open reading frame for dex was 4,011 bp, ending with a stop codon TAA. A ribosome-binding site and putative promoter preceding the start codon were identified. The deduced amino acid sequence of Dex revealed the presence of a signal peptide of 30 amino acids. The cleavage site for the signal sequence was determined by N-terminal amino acid sequence analysis for Dex produced in E. coli chi 2831(pYA902). The C terminus consists of a serine- and threonine-rich region followed by the peptide LPKTGD, 3 charged amino acids, 19 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the cell wall-spanning region, the LPXTGX consensus sequence, and the membrane-anchoring domains of surface-associated proteins of gram-positive cocci.

Amino Acid Sequence↗
PubMed2001

Evidence against an Interaction between the mRNA downstream box and 16S rRNA in translation initiation.

Based on the complementarity of the initial coding region (downstream box [db]) of several bacterial and phage mRNAs to bases 1469 to 1483 in helix 44 of 16S rRNA (anti-downstream box [adb]), it has been proposed that db-adb base pairing enhances translation in a way that is similar to that of the Shine-Dalgarno (SD)/anti-Shine-Dalgarno (aSD) interaction. Computer modeling of helix 44 on the 30S subunit shows that the topography of the 30S ribosome does not allow a simultaneous db-adb interaction and placement of the initiation codon in the ribosomal P site. Thus, the db-adb interaction cannot substitute for the SD-aSD interaction in translation initiation. We have always argued that any contribution of the db-adb interaction should be most apparent on mRNAs devoid of an SD sequence. Here, we show that 30S ribosomes do not bind to leaderless mRNA in the absence of initiator tRNA, even when the initial coding region shows a 15-nucleotide complementarity (optimal fit) with the putative adb. In addition, an optimized db did not affect the translational efficiency of a leaderless lambda cI-lacZ reporter construct. Thus, the db-adb interaction can hardly serve as an initial recruitment signal for ribosomes. Moreover, we show that different leaderless mRNAs are translated in heterologous systems although the sequence of the putative adb's within helix 44 of the 30S subunits of the corresponding bacteria differ largely. Taken our data together with those of others (M. O'Connor, T. Asai, C. L. Squires, and A. E. Dahlberg, Proc. Natl. Acad. Sci. USA 96:8973-8978, 1999; A. La Teana, A. Brandi, M. O'Connor, S. Freddi, and C. L. Pon, RNA 6:1393-1402, 2000), we conclude that the db does not base pair with the adb.

Base Pairing↗
PubMed2003

Refining the relation between 'first hits' and 'second hits' at the APC locus: the 'loose fit' model and evidence for differences in somatic mutation spectra among patients.

The site of the 'first hit' in the APC tumour suppressor gene determines the type of the 'second hit', both in familial adenomatous polyposis (FAP) and sporadic colorectal tumours. Mutations near codon 1300 are associated with loss of heterozygosity (LOH) of the wild-type allele; other tumours tend to have two protein-truncating mutations. In this study, we have confirmed and refined the LOH-associated region in colorectal FAP: allelic loss in adenomatous polyps tended to occur when the germline mutation lay in the region of the APC gene between the first and second beta-catenin degradation repeats (codons 1285-1378). LOH generally occurred by mitotic recombination, leaving two identical alleles, each encoding a protein with one remaining beta-catenin degradation repeat. For patients with germline mutations that truncated the protein before the first repeat (codon 1264), LOH was very rare and tumours generally acquired a somatic mutation which left two, or less often one, repeats remaining in the protein. In our sample set, patients with germline mutations after the second beta-catenin degradation repeat tended to have undetectable, presumably cryptic, somatic mutations in their polyps. Exceptions to these rules were, however, not uncommon. Although the site of the germline mutation was the strongest determinant of the somatic mutation in FAP tumours and most patients showed no clear tendency to acquire specific types of truncating 'second hit', a minority of patients did have unusual somatic mutation spectra in their polyps. Thus, some individuals may be predisposed to particular types of 'second hit' (for example, frameshift rather than nonsense changes). Overall, disease severity (polyp number) did not vary with individuals' spectrum of somatic APC mutations, providing no clear evidence for modifier genes that influence disease severity in this fashion. Our data are consistent with the hypothesis that there exists an optimal level of beta-catenin signalling in colorectal tumours and that the APC mutation spectrum principally reflects this fact. The association between 'first hits' and 'second hits' at APC is not, however, so strong as to suggest that tumorigenesis only occurs if the genotype is optimum; we suggest 'relaxed' terminology, the 'loose fit' model, to describe this situation.

Adenomatous Polyposis Coli↗