Prevalence of faecal excretion of verocytotoxigenic Escherichia coli O157 in cattle in England and Wales.

During the decade to 1999, the incidence of human infections with the zoonotic pathogen verocytotoxin-producing Escherichia coli O157 (VTEC O157) increased in England and Wales. This paper describes the results of a survey of 75 farms to determine the prevalence of faecal excretion of VTEC O157 by cattle, its primary reservoir host, in England and Wales. Faecal samples were collected from 4663 cattle between June and December 1999. The prevalence of excretion by individual cattle was 4.2 per cent (95 per cent confidence interval [CI] 2.0 to 6.4) and 10.3 per cent (95 per cent CI 5.8 to 14.8) among animals in infected herds. The within-herd prevalence on positive farms ranged from 1.1 to 51.4 per cent. At least one positive animal was identified on 29 (38.7 per cent; 95 per cent CI 28.1 to 50.4) of the farms, including dairy, suckler and fattening herds. The prevalence of excretion was least in the calves under two months of age, peaked in the calves aged between two and six months and declined thereafter. The phage types identified most widely were 4, 34 and 2, which were each found on six of the 29 positive farms.

Investigation of the genetic diversity among isolates of Salmonella enterica serovar Dublin from animals and humans from England, Wales and Ireland.

AIMS: To assess the degree of genetic diversity among animal Salmonella Dublin UK isolates, and to compare it with the genetic diversity found among human isolates from the same time period. METHODS AND RESULTS: One hundred isolates (50 human and 50 animal) were typed using plasmid profiling, XbaI-pulsed field gel electrophoresis (PFGE) and PstI-SphI ribotyping. Antimicrobial resistance data to 16 antibiotics was presented, and the presence of class-I integrons was investigated by real-time PCR. Seven different plasmid profiles, 19 ribotypes and 21 PFGE types were detected. A combination of the three methods allowed clear differentiation of 43 clones or strains. Eighteen isolates were resistant to at least one antimicrobial; five of them were multi-resistant and of these, only three presented class I integrons. CONCLUSIONS: Ribotyping data suggest the existence of at least three very different clonal lines; the same distribution in well-defined groups was not evident from the PFGE data. The existence of a variety of clones in both animals and humans has been demonstrated. A few prevalent clones seem to be widely disseminated among different animal species and show a diverse geographical and temporal distribution. The same clones were found in animals and humans, which may infer that both farm and pet animals may act as potential vehicles of infection for humans. Some other clones seem to be less widely distributed. Clustering analysis of genomic fingerprints of Salmonella Dublin and Salm. Enteritidis isolates confirms the existence of a close phylogenetic relationship between both serotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper describes the utility of a multiple genetic typing approach for Salm. Dublin. It gives useful information on clonal diversity among human and animal isolates.

Faecal carriage of verocytotoxin-producing Escherichia coli O157 in cattle and sheep at slaughter in Great Britain.

A 12-month abattoir survey was conducted between January 1999 and January 2000, to determine the prevalence of faecal carriage of verocytotoxin-producing Escherichia coli O157 (VTEC O157) in cattle and sheep slaughtered for human consumption in Great Britain. Samples of rectum containing faeces were collected from 3939 cattle and 4171 sheep at 118 abattoirs, in numbers proportional to the throughput of the premises. The annual prevalence of faecal carriage of VTEC O157 was 4.7 per cent (95 per cent confidence interval 4.1 to 5.4) for cattle and 1.7 per cent (1.3 to 2.1) for sheep, values which were statistically significantly different from each other (P < 0.001). The organisms were recovered from both cattle and sheep slaughtered throughout the year and at abattoirs in all regions of the country, but the highest prevalence was in the summer. The most frequency recovered VTEC O157 isolates were phage types 2, 8 and 21/28 in cattle and 4 and 32 in sheep, the five most frequently isolated phage types associated with illness in people in Great Britain during the same period.

A 5' Leader of Rbm3, a Cold Stress-induced mRNA, Mediates Internal Initiation of Translation with Increased Efficiency under Conditions of Mild Hypothermia.

Although mild hypothermia generally reduces protein synthesis in mammalian cells, the expression of a small number of proteins, including Rbm3, is induced under these conditions. In this study, we identify an Rbm3 mRNA with a complex 5' leader sequence containing multiple upstream open reading frames. Although these are potentially inhibitory to translation, monocistronic reporter mRNAs containing this leader were translated relatively efficiently. In addition, when tested in the intercistronic region of dicistronic mRNAs, this leader dramatically enhanced second cistron translation, both in transfected cells and in cell-free lysates, suggesting that the Rbm3 leader mediates cap-independent translation via an internal ribosome entry site (IRES). Inasmuch as Rbm3 mRNA and protein levels are both increased in cells exposed to mild hypothermia, the activity of this IRES was evaluated at a cooler temperature. Compared to 37 degrees C, IRES activity at 33 degrees C was enhanced up to 5-fold depending on the cell line. Moderate enhancements also occurred with constructs containing other viral and cellular IRESes. These effects of mild hypothermia on translation were not caused by decreased cell growth, as similar effects were not observed when cells were serum starved. The results suggest that cap-independent mechanisms may facilitate the translation of particular mRNAs during mild hypothermia.

Identification of two short internal ribosome entry sites selected from libraries of random oligonucleotides.

Sequences that control translation of mRNA may play critical roles in regulating protein levels. One such element is the internal ribosome entry site (IRES). We previously showed that a 9-nt segment in the 5' leader sequence of the mRNA encoding Gtx homeodomain protein could function as an IRES. To identify other short sequences with similar properties, we designed a selection procedure that uses a retroviral vector to express dicistronic mRNAs encoding enhanced green and cyan fluorescent proteins as the first and second cistrons, respectively. Expression of the second cistron was dependent upon the intercistronic sequences and was indicative of IRES activity. B104 cells were infected with two retroviral libraries that contained random sequences of 9 or 18 nt in the intercistronic region. Cells expressing both cistrons were sorted, and sequences recovered from selected cells were reassayed for IRES activity in a dual luciferase dicistronic mRNA. Two novel IRESes were identified by this procedure, and both contained segments with complementarity to 18S rRNA. When multiple copies of either segment were linked together, IRES activities were dramatically enhanced. Moreover, these synthetic IRESes were differentially active in various cell types. These properties are similar to those of the previously identified 9-nt IRES module from Gtx mRNA. These results provide further evidence that short nucleotide sequences can function as IRESes and support the idea that some cellular IRESes may be composed of shorter functional modules. The ability to identify IRES modules with specific expression properties may be useful in the design of vectors for biotechnology and gene therapy.

Internal initiation of translation of five dendritically localized neuronal mRNAs.

In neurons, translation of dendritically localized mRNAs is thought to play a role in affecting synaptic efficacy. Inasmuch as components of the translation machinery may be limiting in dendrites, we investigated the mechanisms by which translation of five dendritically localized mRNAs is initiated. The 5' leader sequences of mRNAs encoding the activity-regulated cytoskeletal protein, the alpha subunit of calcium-calmodulin-dependent kinase II, dendrin, the microtubule-associated protein 2, and neurogranin (RC3) were evaluated for their ability to affect translation in the 5' untranslated region of a monocistronic reporter mRNA. In both neural and nonneural cell lines, the activity-regulated cytoskeletal protein, microtubule-associated protein 2, and alpha-CaM Kinase II leader sequences enhanced translation, whereas the dendrin and RC3 5' untranslated regions slightly inhibited translation as compared with controls. When cap-dependent translation of these constructs was suppressed by overexpression of a protein that binds the cap-binding protein eIF4E, it was revealed that translation of these mRNAs had both cap-dependent and cap-independent components. The cap-independent component was further analyzed by inserting the 5' leader sequences into the intercistronic region of dicistronic mRNAs. All five leader sequences mediated internal initiation via internal ribosome entry sites (IRESes). The RC3 IRES was most active and was further characterized after transfection in primary neurons. Although translation mediated by this IRES occurred throughout the cell, it was relatively more efficient in dendrites. These data suggest that IRESes may increase translation efficiency at postsynaptic sites after synaptic activation.

Expression of oestrogen receptor alpha variants in non-malignant breast and early invasive breast carcinomas.

Oestrogen receptor (ER) alpha variants have been described in normal breast and breast carcinomas, but their presence in a range of benign conditions and in small early invasive breast carcinomas has not been considered. Cryostat tissue sections from 19 normal and proliferative breast lesions and 44 carcinomas 15 mm and less in size detected by mammographic screening were screened for ERalpha splice variants using reverse transcriptase-nested PCR. The carcinomas were assessed for mutation by single-stranded conformational polymorphism analysis and variant forms/band shifts were sequenced. ERalpha was detected in all 19 non-malignant cases and exon 7-deleted variants were found in 16 of them. Three cases showed weak expression of exon 5, and two of exon 3 variants. There was no relationship between the presence of variants and the extent of proliferative change, ER status or age. ERalpha mRNA was not detected in two carcinomas; exon 3 deletions were found in four (9. 5%) of the other carcinomas, exon 5 in two (4.8%), and exon 7 in 11 (26.2%), with two variants in four carcinomas and a total of 29.5% of all cases having detectable variants. Two point mutations were found in one, which was a tubular carcinoma. Variant forms were identified in carcinomas of all sizes (bar<10 mm) but were more frequent in those of 15 mm. There was no relationship with type, grade or receptor status. The main difference between non-malignant breast and early invasive cancers related to exons 3 and 5. The findings suggest that ERalpha variants are not involved in breast cancer development but occur with tumour progression and may be a consequence rather than a cause.

A mutation in the c-myc-IRES leads to enhanced internal ribosome entry in multiple myeloma: a novel mechanism of oncogene de-regulation.

The 5' untranslated region of the proto-oncogene c-myc contains an internal ribosome entry segment (IRES) (Nanbru et al., 1997; Stoneley et al., 1998) and thus c-myc protein synthesis can be initiated by a cap-independent as well as a cap-dependent mechanism (Stoneley et al., 2000). In cell lines derived from patients with multiple myeloma (MM) there is aberrant translational regulation of c-myc and this correlates with a C-T mutation in the c-myc-IRES (Paulin et al., 1996). RNA derived from the mutant IRES displays enhanced binding of protein factors (Paulin et al., 1998). Here we show that the same mutation is present in 42% of bone marrow samples obtained from patients with MM, but was not present in any of 21 controls demonstrating a strong correlation between this mutation and the disease. In a tissue culture based assay, the mutant version of the c-myc-IRES was more active in all cell types tested, but showed the greatest activity in a cell line derived from a patient with MM. Our data demonstrate that a single mutation in the c-myc-IRES is sufficient to cause enhanced initiation of translation via internal ribosome entry and represents a novel mechanism of oncogenesis.

A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity.

This study addresses the properties of a newly identified internal ribosome entry site (IRES) contained within the mRNA of the homeodomain protein Gtx. Sequential deletions of the 5' untranslated region (UTR) from either end did not define distinct IRES boundaries; when five nonoverlapping UTR fragments were tested, four had IRES activity. These observations are consistent with other cellular IRES analyses suggesting that some cellular IRESes are composed of segments (IRES modules) that independently and combinatorially contribute to overall IRES activity. We characterize a 9-nt IRES module from the Gtx 5' UTR that is 100% complementary to the 18S rRNA at nucleotides 1132-1124. In previous work, we demonstrated that this mRNA segment could be crosslinked to its complement within intact 40S subunits. Here we show that increasing the number of copies of this IRES module in the intercistronic region of a dicistronic mRNA strongly enhances IRES activity in various cell lines. Ten linked copies increased IRES activity up to 570-fold in Neuro 2a cells. This level of IRES activity is up to 63-fold greater than that obtained by using the well characterized encephalomyocarditis virus IRES when tested in the same assay system. When the number of nucleotides between two of the 9-nt Gtx IRES modules was increased, the synergy between them decreased. In light of these findings, we discuss possible mechanisms of ribosome recruitment by cellular mRNAs, address the proposed role of higher order RNA structures on cellular IRES activity, and suggest parallels between IRES modules and transcriptional enhancer elements.

c-Myc protein synthesis is initiated from the internal ribosome entry segment during apoptosis.

Recent studies have shown that during apoptosis protein synthesis is inhibited and that this is in part due to the proteolytic cleavage of eukaryotic initiation factor 4G (eIF4G). Initiation of translation can occur either by a cap-dependent mechanism or by internal ribosome entry. The latter mechanism is dependent on a complex structural element located in the 5' untranslated region of the mRNA which is termed an internal ribosome entry segment (IRES). In general, IRES-mediated translation does not require eIF4E or full-length eIF4G. In order to investigate whether cap-dependent and cap-independent translation are reduced during apoptosis, we examined the expression of c-Myc during this process, since we have shown previously that the 5' untranslated region of the c-myc proto-oncogene contains an IRES. c-Myc expression was determined in HeLa cells during apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand. We have demonstrated that the c-Myc protein is still expressed when more than 90% of the cells are apoptotic. The presence of the protein in apoptotic cells does not result from either an increase in protein stability or an increase in expression of c-myc mRNA. Furthermore, we show that during apoptosis initiation of c-myc translation occurs by internal ribosome entry. We have investigated the signaling pathways that are involved in this response, and cotransfection with plasmids which harbor either wild-type or constitutively active MKK6, a specific immediate upstream activator of p38 mitogen-activated protein kinase (MAPK), increases IRES-mediated translation. In addition, the c-myc IRES is inhibited by SB203580, a specific inhibitor of p38 MAPK. Our data, therefore, strongly suggest that the initiation of translation via the c-myc IRES during apoptosis is mediated by the p38 MAPK pathway.

Microsatellite instability in ductal carcinoma in situ of the breast.

Microsatellite instability (MI+) is associated with defects in mismatch repair, resulting in a 'mutator' phenotype and the development and progression of cancer. MI+ has been documented in invasive breast carcinomas. This study was undertaken to determine whether MI+ is found in the early non-invasive form of breast cancer, ductal carcinoma in situ (DCIS). We examined microdissected ducts from 23 cases of DCIS with 11 markers comprising mono-, di-, and trinucleotide repeats from six chromosomal regions. Five tumours (22 per cent) displayed MI+ at two or more loci, in all ducts examined. A further seven (30 per cent) tumours showed alterations at a single locus (the DM-1 trinucleotide), and for two of these, heterogeneity between ducts was observed. Alterations at microsatellite repeat motifs in the coding regions of four cancer-associated genes (TGF beta RII, IGFIIR, BAX, and E2F-4) were not observed. Immunohistochemistry revealed that there was no loss of reactivity for the mismatch repair proteins, MLH1, MSH2, and PMS2, in the DCIS cases. In general, MI+ tumours and those with alterations at the DM-1 microsatellite were predominantly of higher nuclear grade and expressing c-erbB-2, suggesting that aberrations in DNA repair functions may lead to the acquisition of a more aggressive phenotype in breast cancer.

A single nucleotide change in the c-myc internal ribosome entry segment leads to enhanced binding of a group of protein factors.

A 340 nucleotide section of the c- myc 5' untranslated region (UTR) contains an internal ribosome entry segment. We have described previously a mutation in this region of RNA in cell lines derived from patients with multiple myeloma (MM) which exhibit increased expression of c- myc protein by an aberrant translational mechanism. In this study we show by electrophoretic mobility shift assays (EMSA), north-western blotting and UV cross-linking that radiolabelled c- myc 5' UTR RNA transcripts which harbour the mutation cause enhanced binding of cellular proteins. In addition, we also demonstrate that an MM derived cell line possesses an altered repertoire of RNA binding proteins. Our data suggest that the deregulated expression of c -myc in MM could result both from the effect of the mutation and the additional proteins which are present in these cell types.

C-Myc 5' untranslated region contains an internal ribosome entry segment.

Translation in eukaryotic cells is generally initiated by ribosome scanning from the 5' end of the capped mRNA. However, initiation of translation may also occur by a mechanism which is independent of the cap structure and in this case ribosomes are directed to the start codon by an internal ribosome entry segment (IRES). Picornaviruses represent the paradigm for this mechanism, but only a few examples exist which show that this mechanism is used by eukaryotic cells. In this report we show data which demonstrate that the 5' UTR of the proto-oncogene c-myc contains an IRES. When a dicistronic reporter vector, with c-myc 5' UTR inserted between the two cistrons, was transfected into both HepG2 and HeLa cells, the translation of the downstream cistron was increased by 50-fold, demonstrating that translational regulation of c-myc is mediated through cap-independent mechanisms. This is the first example of a proto-oncogene regulated in this manner and suggests that aberrant translational regulation of c-myc is likely to play a role in tumorigenesis.

Loss of heterozygosity at chromosome 6q in preinvasive and early invasive breast carcinomas.

We have used polymerase chain reaction (PCR) analysis to study the incidence of allelic imbalance at four polymorphic microsatellite markers on chromosome 6q25.1-27, three dinucleotide repeats and one trinucleotide repeat, for microdissected tumour foci from a group of 75 'early' breast carcinomas. The tumours comprised 16 preinvasive cases of ductal carcinoma in situ (DCIS) and 59 mammographically detected early invasive carcinomas. Loss of heterozygosity (LOH) was detected at all four loci and in all types and grade of disease. The frequency of LOH ranged from 23% to 50% depending on the marker studied. The highest frequency of LOH was observed at the D6S186 locus for the cases of DCIS and at the oestrogen receptor locus for the invasive carcinomas. These data suggest that the inactivation of tumour-suppressor genes within this region on chromosome 6q is important for the development of these early lesions.

Loss of heterozygosity at the mannose 6-phosphate insulin-like growth factor 2 receptor gene correlates with poor differentiation in early breast carcinomas.

Chromosome 6q has been shown to be one of the most frequent sites for allelic loss in human breast cancer. The mannose 6-phosphate/insulin-like growth factor 2 receptor (IGF2R) gene, which maps to chromosome 6q26-27, functions in the activation of TGF-beta1, a potent growth inhibitor for most cell types, the degradation of the mitogen IGF2 and the intracellular trafficking of lysosomal enzymes. Loss of heterozygosity (LOH) at the IGF2R locus with mutations in the remaining allele have been reported in liver cancers and recently in two high-grade cases of ductal carcinoma in situ of the breast. We have sought to confirm that allelic loss of IGF2R is an early event in the aetiology of breast cancer by screening a group of 'early' lesions for LOH at a polymorphic microsatellite marker within the IGF2R gene using polymerase chain reaction (PCR). Several microdissected tumour foci were analysed for each of 40 mammographically detected invasive carcinomas and 22 cases of pure ductal carcinoma in situ (DCIS). None of 25 (62.5%) informative early invasive carcinomas showed any evidence of LOH. This group comprised predominantly of well- to moderately differentiated cases (95%). However, 4 out of 18 informative DCIS cases (22%) showed clear evidence of LOH. Three of these were poorly differentiated (high-grade) lesions. These data suggest that loss of heterozygosity at the IGF2R gene is associated with poor differentiation at this early stage of breast cancer development and progression.

Microsatellite instability in early sporadic breast cancer.

We have studied the incidence of microsatellite instability at three trinucleotide repeats and seven dinucleotide repeats from five chromosomal regions, in a group of 30 mammographically detected 'early' invasive breast cancers and correlated its occurrence with clinicopathological parameters. The myotonic dystrophy (DM-1) trinucleotide repeat was analysed in 48 additional cases. In 4 out of 78 (5%) paired tumour-normal DNA samples we found evidence of somatic microsatellite instability at DM-1: a novel allele of a different size was seen in the tumour DNA which was not present in the normal DNA sample. All four tumours that showed evidence of instability were from the core group of 30 cases (13%) and were well or moderately differentiated, oestrogen receptor-positive, infiltrating ductal carcinomas. Two of these tumours were unstable at nine of ten loci studied, both trinucleotide and dinucleotide repeats. DNA prepared from different normal tissues showed no evidence of instability, for all four instability cases. These data indicate that microsatellite instability is specific to the tumour DNA and is an early event in the genesis of some sporadic breast cancers.