Absolute quantitative real-time polymerase chain reaction for the measurement of human papillomavirus E7 mRNA in cervical cytobrush specimens.

BACKGROUND: Few reports of the utilization of an accurate, cost-effective means for measuring HPV oncogene transcripts have been published. Several papers have reported the use of relative quantitation or more expensive Taqman methods. Here, we report a method of absolute quantitative real-time PCR utilizing SYBR-green fluorescence for the measurement of HPV E7 expression in cervical cytobrush specimens. RESULTS: The construction of a standard curve based on the serial dilution of an E7-containing plasmid was the key for being able to accurately compare measurements between cervical samples. The assay was highly reproducible with an overall coefficient of variation of 10.4%. CONCLUSION: The use of highly reproducible and accurate SYBR-based real-time polymerase chain reaction (PCR) assays instead of performing Taqman-type assays allows low-cost, high-throughput analysis of viral mRNA expression. The development of such assays will help in refining the current screening programs for HPV-related carcinomas.

Towards the identification of a tumor suppressor gene on chromosome 3p12: Physical mapping and candidate gene screening

Renal cell carcinoma (RCC) is the most common malignant tumor of the kidney. Characterization of RCC tumors indicates that the most frequent genetic event associated with the initiation of tumor formation involves a loss of heterozygosity or cytogenetic aberration on the short arm of human chromosome 3. A tumor suppressor locus Nonpapillary Renal Carcinoma-1 (NRC-1, OMIM ID 604442) has been previously mapped to a 5–7 cM region on chromosome 3p12 and shown to induce rapid tumor cell death in vivo, as demonstrated by functional complementation experiments. ^ To identify the gene that accounts for the tumor suppressor activities of NRC-1, fine-scale physical mapping was conducted with a novel real-time quantitative PCR based method developed in this study. As a result, NRC-1 was mapped within a 4.6-Mb region defined by two unique sequences within UniGene clusters Hs.41407 and Hs.371835 (78,545Kb–83,172Kb in the NCBI build 31 physical map). The involvement of a putative tumor suppressor gene Robo1/Dutt1 was excluded as a candidate for NRC-1. Furthermore, a transcript map containing eleven candidate genes was established for the 4.6-Mb region. Analyses of gene expression patterns with real-time quantitative RT-PCR assays showed that one of the eleven candidate genes in the interval (TSGc28) is down-regulated in 15 out of 20 tumor samples compared with matched normal samples. Three exons of this gene have been identified by RACE experiments, although additional exon(s) seem to exist. Further gene characterization and functional studies are required to confirm the gene as a true tumor suppressor gene. ^ To study the cellular functions of NRC-1, gene expression profiles of three tumor suppressive microcell hybrids, each containing a functional copy of NRC-1, were compared with those of the corresponding parental tumor cell lines using 16K oligonucleotide microarrays. Differentially expressed genes were identified. Analyses based on the Gene Ontology showed that introduction of NRC-1 into tumor cell lines activates genes in multiple cellular pathways, including cell cycle, signal transduction, cytokines and stress response. NRC-1 is likely to induce cell growth arrest indirectly through WEE1. ^

LDOC1, A NOVEL BIOMARKER OF PROGNOSIS IN CHRONIC LYMPHOCYTIC LEUKEMIA

In chronic lymphocytic leukemia (CLL), one of the best predictors of outcome is the somatic mutation status of the immunoglobulin heavy chain variable region (IGHV) genes. Patients whose CLL cells have unmutated IGHV genes have a median survival of 8 years; those with mutated IGHV genes have a median survival of 25 years. To identify new prognostic biomarkers and molecular targets for therapy in untreated CLL patients, we reanalyzed the raw data from four published gene expression profiling microarray studies. Of 88 candidate biomarkers associated with IGHV somatic mutation status, we identified LDOC1 (Leucine Zipper, Down-regulated in Cancer 1), as one of the most significantly differentially expressed genes that distinguished mutated from unmutated CLL cases. LDOC1 is a putative transcription factor of unknown function in B-cell development and CLL pathophysiology. Using a highly sensitive quantitative RT-PCR (QRT-PCR) assay, we confirmed that LDOC1 mRNA was dramatically down-regulated in mutated compared to unmutated CLL cases. Expression of LDOC1 mRNA was also vii strongly associated with other markers of poor prognosis, including ZAP70 protein and cytogenetic abnormalities of poor prognosis (deletions of chromosomes 6q21, 11q23, and 17p13.1, and trisomy 12). CLL cases positive for LDOC1 mRNA had significantly shorter overall survival than negative cases. Moreover, in a multivariate model, LDOC1 mRNA expression predicted overall survival better than IGHV mutation status or ZAP70 protein, among the best markers of prognosis in CLL. We also discovered LDOC1S, a new LDOC1 splice variant. Using isoform-specific QRT-PCR assays that we developed, we found that both isoforms were expressed in normal B cells (naïve > memory), unmutated CLL cells, and in B-cell non-Hodgkin lymphomas with unmutated IGHV genes. To investigate pathways in which LDOC1 is involved, we knocked down LDOC1 in HeLa cells and performed global gene expression profiling. GFI1 (Growth Factor-Independent 1) emerged as a significantly up-regulated gene in both HeLa cells and CLL cells that expressed high levels of LDOC1. GFI1 oncoprotein is implicated in hematopoietic stem cell maintenance, lymphocyte development, and lymphomagenesis. Our findings indicate that LDOC1 mRNA is an excellent biomarker of overall survival in CLL, and may contribute to B-cell differentiation and malignant transformation.

IDENTIFICATION OF FACTORS INVOLVED IN DNA METHYLATION OF CPG-ISLAND-PROMOTERS

Repression of many tumor suppressor genes (TSGs) in cancer is mediated by aberrantly increased DNA methylation levels at promoter CpG islands (CGI). About one-fourth of empirically defined human promoters are surrounded by or contain clustered repetitive elements. It was previously observed that a sharp transition of methylation occurs between highly methylated repetitive elements (SINE or LINE) and unmethylated CGI-promoters (e.g. P16, VHL, CDH and RIL) in normal tissues. The functions that lead to increased CGI methylation in cancer remain poorly understood. We propose that CGI-promoters contain cis-elements for triggering de novo DNA methylation. In the first part of our project, we established a site-specific integration system with enforced local transcriptional repression in colorectal cancer cells and monitored the occurrence of de novo DNA methylation in exogenous fragments containing a CGI-promoter and repetitive elements. Initial de novo methylation was seeded at specific CG sites in a repetitive element, and accelerated by persistent binding of a KRAB-containing transcriptional repressor. Furthermore, additional repetitive elements (LINE and SINE) located adjacent to the promoter could confer DNA methylation spreading into the CGI particularly in the setting of KRAB-factor binding. However, a repressive chromatin alone was not sufficient to initiate DNA methylation, which required specific DNA sequences and was integration-site (and/or cell-line) specific. In addition, all the methylation observed showed slow and gradual accumulation over several months of culture. Overall, these results demonstrate a requirement for specific DNA sequences to trigger de novo DNA methylation, and repetitive elements as cis-regulatory factors to cooperate with strengthened transcriptional repression in promoting methylation spreading. In the second part, we re-introduced disrupted DNMT3B or DNMT1 into HCT116 DKO cells and mapped the remethylation pattern through a profiling method (DREAM). Moderate remethylation occurred when DNMT3B was re-expressed with a preference toward non-CGI and non-promoter regions. Hence, there exists a set of genomic regions with priority to be targets for DNMT3B in somatic cells.

Prognostic significance of xCT polymorphisms and expression in patients with advanced pancreatic cancer treated with chemotherapy

The plasma membrane xc- cystine/glutamate transporter mediates cellular uptake of cystine in exchange for intracellular glutamate and is highly expressed by pancreatic cancer cells. The xCT gene, encoding the cystine-specific xCT protein subunit of xc-, is important in regulating intracellular glutathione (GSH) levels, critical for cancer cell protection against oxidative stress, tumor growth and resistance to chemotherapeutic agents including platinum. We examined 4 single nucleotide polymorphisms (SNPs) of the xCT gene in 269 advanced pancreatic cancer patients who received first line gemcitabine with or without cisplatin or oxaliplatin. Genotyping was performed using Taqman real-time PCR assays. A statistically significant correlation was noted between the 3' untranslated region (UTR) xCT SNP rs7674870 and overall survival (OS): Median survival time (MST) was 10.9 and 13.6 months, respectively, for the TT and TC/CC genotypes (p = 0.027). Stratified analysis showed the genotype effect was significant in patients receiving gemcitabine in combination with platinum therapy (n = 145): MST was 10.5 versus 14.1 months for the TT and TC/CC genotypes, respectively (p = 0.013). The 3' UTR xCT SNP rs7674870 may correlate with OS in pancreatic cancer patients receiving gemcitabine and platinum combination therapy. Paraffin-embedded core and surgical biopsy tumor specimens from 98 patients with metastatic pancreatic adenocarcinoma were analyzed by immunohistochemistry using an xCT specific antibody. xCT protein IHC expression scores were analyzed in relation to overall survival in 86 patients and genotype in 12 patients and no statistically significant association was found between the level of xCT IHC expression score and overall survival (p = 0.514). When xCT expression was analyzed in terms of treatment response, no statistically significant associations could be determined (p = 0.908). These data suggest that polymorphic variants of xCT may have predictive value, and that the xc- transporter may represent an important target for therapy in pancreatic cancer.

Cloning, characterization and regulation of the soluble guanylyl cyclase alpha1 and beta1 genes

Cell signaling by nitric oxide (NO) through soluble guanylyl cyclase (sGC) and cGMP production regulates physiological responses such as smooth muscle relaxation, neurotransmission, and cell growth and differentiation. Although the NO receptor, sGC, has been studied extensively at the protein level, information on regulation of the sGC genes remains elusive. In order to understand the molecular mechanisms involved at the level of gene expression, cDNA and genomic fragments of the murine sGCα1 subunit gene were obtained through library screenings. Using the acquired clones, the sGCα 1 gene structure was determined following primer extension, 3 ′RACE and intron/exon boundary analyses. The basal activity of several 5′-flanking regions (putative promoter regions) for both the α1 and β1 sGC subunits were determined following their transfection into mouse N1E-115 neuroblastoma and rat RENE1Δ14 uterine epithelial cells using a luciferase reporter plasmid. Using the sGC sequences, real-time RT-PCR assays were designed to measure mRNA levels of the sGC α1 and β1 genes in rat, mouse and human. Subsequent studies found that uterine sGC mRNA and protein levels decreased rapidly in response to 17β-estradiol (estrogen) in an in vivo rat model. As early as 1 hour following treatment, mRNA levels of both sGC mRNAs decreased, and reached their lowest level of expression after 3 hours. This in vivo response was completely blocked by the pure estrogen receptor antagonist, ICI 182,780, was not seen in several other tissues examined, did not occur in response to other steroid hormones, and was due to a post-transcriptional mechanism. Additional studies ex vivo and in various cell culture models suggested that the estrogen-mediated decreased sGC mRNA expression did not require signals from other tissues, but may require cell communication or paracrine factors between different cell types within the uterus. Using chemical inhibitors and molecular targeting in other related studies, it was revealed that c-Jun-N-terminal kinase (JNK) signaling was responsible for decreased sGC mRNA expression in rat PC12 and RFL-6 cells, two models previously determined to exhibit rapid decreased sGC mRNA expression in response to different stimuli. To further investigate the post-transcriptional gene regulation, the full length sGCα1 3′-untranslated region (3′UTR) was cloned from rat uterine tissue and ligated downstream of the rabbit β-globin gene and expressed as a chimeric mRNA in the rat PC12 and RFL-6 cell models. Expression studies with the chimeric mRNA showed that the sGCα 1 3′UTR was not sufficient to mediate the post-transcriptional regulation of its mRNA by JNK or cAMP signaling in PC12 and RFL-6 cells. This study has provided numerous valuable tools for future studies involving the molecular regulation of the sGC genes. Importantly, the present results identified a novel paradigm and a previously unknown signaling pathway for sGC mRNA regulation that could potentially be exploited to treat diseases such as uterine cancers, neuronal disorders, hypertension or various inflammatory conditions. ^

POU domain transcription factor Brn3b is critical for the normal development and survival of retinal ganglion cells

The POU domain transcription factor Brn3b/POU4F2 plays a critical role regulating gene expression in mouse retinal ganglion cells (RGCs). Previous investigations have shown that Brn3b is not required for initial cell fate specification or migration; however, it is essential for normal RGC differentiation. In contrast to wild type axons, the mutant neurites were phenotypically different: shorter, rougher, disorganized, and poorly fasciculated. Wild type axons stained intensely with axon specific marker tau-1, while mutant projections were weakly stained and the mutant projections showed strong labeling with dendrite specific marker MAP2. Brn-3b mutant axonal projections contained more microtubules and fewer neurofilaments, a dendritic characteristic, than the wild type. The mutant neurites also exhibited significantly weaker staining of neurofilament low-molecular-weight (NF-L) in the axon when compared to the wild type, and NF-L accumulation in the neuron cell body. The absence of Brn-3b results in an inability to form normal axons and enhanced apoptosis in RGCs, suggesting that Brn-3b may control a set of genes involved in axon formation. ^ Brn3b contains several distinct sequence motifs: a glycine/serine rich region, two histidine rich regions, and a fifteen amino acid conserved sequence shared by all Brn3 family members in the N-terminus and a POU specific and POU homeodomain in the C-terminus. Brn3b activates a Luciferase reporter over 25 fold in cell culture when binding to native brn3 binding sites upstream of a minimal promoter. When fused to the Gal4 DNA Binding domain (DBD) and driven by either a strong (CMV) or weaker (pAHD) promoter, the N-terminal of Brn3b is capable of similar activation when binding to Gal4 UAS sites, indicating a presumptive activator of transcription. Both full length Brn3b or the C-terminus fused to the Gal4DBD and driven by pCMV repressed a Luciferase reporter downstream of UAS binding sites. Lower levels of expression of the fusion protein driven by pADH resulted in an alleviation of repression. This repression appears to be a limitation of this system of transcriptional analysis and a potential pitfall in conventional pCMV based transfection assays. ^

Carcinoma of the lower uterine segment: A newly described association with Lynch Syndrome

Purpose. We performed a case-comparison study to describe the characteristics of LUS tumors and their association with risk factors for endometrial cancer. ^ Patients and Methods. From January 1996 through October 2007, 3,892 women were identified with a diagnosis of primary endometrial carcinoma or primary cervical adenocarcinoma. Pathology records from the 1,009 women who had a hysterectomy were reviewed. Subjects were included in the LUS group only if the tumor was clearly originating from the area between the lower corpus and upper cervix in the hysterectomy specimen. The LUS group was compared to all patients with endometrial corpus carcinoma who underwent hysterectomy at our institution in a 12-month period randomly selected from the study period. Risk factors for endometrial carcinoma such as body mass index (BMI) and Lynch Syndrome were assessed. Expression of estrogen receptor (ER), vimentin, carcinoembryonic antigen (CEA), p16, and human papilloma virus DNA (HPV DNA) was assessed; this panel is known to be effective in distinguishing adenocarcinomas of endometrial versus endocervical origin. Fisher's Exact, Chi-square, Mann-Whitney, and Student's t-tests were utilized for statistical analysis. ^ Results. Thirty-five of 1,009 women had endometrial carcinoma of the LUS (3.5%; 95% CI: 2–4%). Compared to patients with corpus tumors, LUS patients were younger (54.2 vs. 62.9 years, P = .001), had higher stage (P < .001), and more invasive tumors (P = .001). Preoperative diagnosis of the LUS tumors more frequently included the possibility of endocervical adenocarcinoma ( P < .001), leading to preoperative radiation therapy in 4 patients. Median BMI was similar in the LUS and corpus groups. Seventy-three percent of the available LUS tumors had a similar immunohistochemical expression pattern to conventional endometrioid adenocarcinoma. Because of the young median age for the LUS group, we performed immunohistochemistry for Lynch syndrome-associated DNA mismatch repair proteins MLH1, MSH2, MSH6, and PMS2. Microsatellite instability testing (MSI) and MLH1 promoter hypermethylation were performed when indicated. Thirty-six percent of the LUS tumors were MSI-high. Ten of thirty-five (29%) women with LUS tumors were either confirmed to have Lynch Syndrome or were strongly suspected to have Lynch Syndrome based on tissue-based molecular assays (95% CI, 16 to 45%). ^ Conclusions. Endometrial carcinoma arising in the LUS is a clinical and pathologic entity which can be diagnostically confused with cervical adenocarcinoma. In general, LUS tumors can be correctly identified as being endometrial carcinoma using the immunohistochemical panel noted above. The prevalence of Lynch Syndrome in patients with LUS tumors is much greater than that of the general endometrial cancer population (1.8%) or in endometrial cancer patients younger than 50 years of age (8–9%). Based on our results, the possibility of Lynch Syndrome should be considered in women with LUS tumors. ^

PCR-based assay using occult blood detection cards for detection of diarrheagenic Escherichia coli in specimens from U.S. travelers to Mexico with acute diarrhea.

Large field studies of travelers' diarrhea for multiple destinations are limited by the need to perform stool cultures on site in a timely manner. A method for the collection, transport, and storage of fecal specimens that does not require immediate processing and refrigeration and that is stable for months would be advantageous. This study was designed to determine if enterotoxigenic Escherichia coli (ETEC) and enteroaggregative E. coli (EAEC) DNA could be identified from cards that were processed for the evaluation of fecal occult blood. U.S. students traveling to Mexico during 2005 to 2007 were monitored for the occurrence of diarrheal illness. When ill, students provided a stool specimen for culture and occult blood by the standard methods. Cards then were stored at room temperature prior to DNA extraction. Fecal PCR was performed to identify ETEC and EAEC in DNA extracted from stools and from occult blood cards. Significantly more EAEC cases were identified by PCR that was performed on DNA that was extracted from cards (49%) or from frozen feces (40%) than from culture methods that used HEp-2 adherence assays (13%) (P < 0.001). Similarly, more ETEC cases were detected from card DNA (38%) than from fecal DNA (30%) or by culture that was followed by hybridization (10%) (P < 0.001). The sensitivity and specificity of the card test were 75 and 62%, respectively, compared to those for EAEC by culture and were 50 and 63%, respectively, compared to those for ETEC. DNA extracted from fecal cards that was used for the detection of occult blood is of use in identifying diarrheagenic E. coli.

A PCR based assay for the detection of enteric pathogens from Hemoccult(RTM) cards

Background. Large field studies in travelers' diarrhea (TD) in multiple destinations are limited by the need to perform stool cultures on site in a timely manner. A method for the collection, transport and storage of fecal specimens that does not require immediate processing, refrigeration and is stable for months would be advantageous. ^ Objectives. Determine if enteric pathogen bacterial DNA can be identified in cards routinely used for evaluation of fecal occult blood. ^ Methods. U.S. students traveling to Mexico in 2005-07 were followed for occurrence of diarrheal illness. When ill, students provided a stool specimen for culture and occult blood by the standard method. Cards were then stored at room temperature prior to DNA extraction. A multiplex fecal PCR was performed to identify enterotoxigenic Escherichia coli and enteroaggregative E. coli (EAEC) in DNA extracted from stools and occult blood cards. ^ Results. Significantly more EAEC cases were identified by PCR done in DNA extracted from cards (49%) or from frozen feces (40%) than by culture followed by HEp-2 adherence assays (13%). Similarly more ETEC cases were detected in card DNA (38%) than fecal DNA (30%) or culture followed by hybridization (10%). Sensitivity and specificity of the card test was 75% and 62%, respectively, and 50% and 63%, respectively, when compared to EAEC and ETEC culture, respectively, and 53% and 51%, respectively compared to EAEC multiplex fecal PCR and 56% and 70%, respectively, compared to ETEC multiplex fecal PCR. ^ Conclusions. DNA extracted from fecal cards used for detection of occult blood is of use in detecting enteric pathogens. ^

MECHANISM-BASED STRATEGIES TO ENHANCE THE ACTIONS OF A

Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the functional stability of client oncoproteins, such as STAT3, Raf-1 and Akt, which play a role in the survival of malignant cells. The chaperone function of HSP90 is driven by the binding and hydrolysis of ATP. The geldanamycin analog, 17-AAG, binds to the ATP pocket of HSP90 leading to the degradation of client proteins. However, treatment with 17-AAG results in the elevation of the levels of antiapoptotic proteins HSP70 and HSP27, which may lead to cell death resistance. The increase in HSP70 and HSP27 protein levels is due to the activation of the transcription factor HSF-1 binding to the promoter region of HSP70 and HSP27 genes. HSF-1 binding subsequently promotes HSP70 and HSP27 gene expression. Based on this, I hypothesized that inhibition of transcription/translation of HSP or client proteins would enhance 17-AAG-mediated cytotoxicity. Multiple myeloma (MM) cell lines MM.1S, RPMI-8226, and U266 were used as a model. To test this hypothesis, two different strategies were used. For the first approach, a transcription inhibitor was combined with 17-AAG. The established transcription inhibitor Actinomycin D (Act D), used in the clinic, intercalates into DNA and blocks RNA elongation. Stress inducible (HSP90á, HSP70 and HSP27) and constitutive (HSP90â and HSC70) mRNA and protein levels were measured using real time RT-PCR and immunoblot assays. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the MM cell lines. This induction of HSP mRNA levels was diminished by 0.05 µg/mL Act D for 12 hours in the combination treatment, except for HSP70. At the protein level, Act D abrogated the 17-AAG-mediated induction of all HSP expression levels, including HSP70. Cytotoxic evaluation (Annexin V/7-AAD assay) of Act D in combination with 17-AAG suggested additive or more than additive interactions. For the second strategy, an agent that affected bioenergy production in addition to targeting transcription and translation was used. Since ATP is necessary for the proper folding and maturation of client proteins by HSP90, ATP depletion should lead to a decrease in client protein levels. The transcription and translation inhibitor 8-Chloro-Adenosine (8-Cl-Ado), currently in clinical trials, is metabolized into its cytotoxic form 8-Cl-ATP causing a parallel decrease of the cellular ATP pool. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the three MM cell lines evaluated. In the combination treatment, 10 µM 8-Cl-Ado for 20 hours did not abrogate the induction of HSP mRNA or protein levels. Since cellular bioenergy is necessary for the stabilization of oncoproteins by HSP90, immunoblot assays analyzing for expression levels of client proteins such as STAT3, Raf-1, and Akt were performed. Immunoblot assays detecting for the phosphorylation status of the translation repressor 4E-BP1, whose activity is modulated by upstream kinases sensitive to changes in ATP levels, were also performed. The hypophosphorylated state of 4E-BP1 leads to translation repression. Data indicated that treatment with 17-AAG alone resulted in a minor (<10%) change in STAT3, Raf-1, and Akt protein levels, while no change was observed for 4E-BP1. The combination treatment resulted in more than 50% decrease of the client protein levels and hypophosphorylation of 4E-BP1 in all MM cell lines. Treatment with 8-Cl-Ado alone resulted in less than 30% decrease in client protein levels as well as a decrease in 4E-BP1 phosphorylation. Cytotoxic evaluation of 8-Cl-Ado in combination with 17-AAG resulted in more than additive cytotoxicity when drugs were combined in a sequential manner. In summary, these data suggest that the mechanism-based combination of agents that target transcription, translation, or decrease cellular bioenergy with 17-AAG results in increase cytotoxicity when compared to the single agents. Such combination strategies may be applied in the clinic since these drugs are established chemotherapeutic agents or currently in clinical trials.

Mapping and characterization of the Xlsirt P11 RNA cis-acting localization element

In many organisms, polarity of the oocyte is established post-transcriptionally via subcellular RNA localization. Many RNAs are localized during oogenesis in Xenopus laevis, including Xlsirts ( Xenopus laevis short interspersed repeat transcripts) [Kloc, 1993]. Xlsirts constitute a large family defined by highly homologous repeat units 79–81 nucleotides in length. Endogenous Xlsirt RNAs use the METRO (Message Transport Organizer) pathway of localization, where RNAs are transported from the nucleus to the mitochondrial cloud in stage I oocytes. Secondly, RNAs anchor at the vegetal pole in stage II oocytes. Exogenous Xlsirt RNAs can also utilize the Late pathway of localization, which involves localization to the vegetal cortex during stage III of oogenesis and results in RNAs anchored in the cortex of the entire vegetal hemisphere. ^ The Xlsirts localization signal is contained within the repeat region. This study was designed to test the hypothesis that there are cis -acting localization elements in Xlsirts, and that higher order structure plays a role. Results of experiments on Xlsirt P11, a 1700 basepair (bp) family member, led to the conclusion that a 137-bp fragment of the repetitive region is necessary and sufficient for METRO and Late pathway localization. This analysis definitively demonstrates that the Xlsirt localization signal for the METRO and Late pathways reside within the repetitive region and not within the flanking regions. Analysis of Xlsirt linker scanning mutations revealed two METRO-pathway specific subelements, and one Late-pathway specific subelement. Functional, computer, and biochemical evidence relates the higher order structure of this element to its ability to function as a localization element. ^ Xlsirt 137 is 99% identical to the Xlsirt consensus sequence identified in this study, suggesting that it is the localization element for all localized Xlsirt family members. The repeat unit was reframed based on function, rather than arbitrarily based on sequence. This work supports the hypothesis presented in 1981 by George Spohr, who originally isolated the Xlsirts, which stated that the highly conserved repetitive elements must be constrained from variability due to some unknown function of the repeats themselves. These studies shed light on the mechanism of RNA localization, linking structure and function. ^