Cross-species comparison illuminates the importance of iron homeostasis for splenic anti-immunosenescence.

Although immunosenescence may result in increased morbidity and mortality, many mammals have evolved effective immune coping strategies to extend their lifespans. Thus, the immune systems of long-lived mammals present unique models to study healthy longevity. To identify the molecular clues of anti-immunosenescence, we first built high-quality reference genome for a long-lived myotis bat, and then compared three long-lived mammals (i.e., bat, naked mole rat, and human) versus the short-lived mammal, mouse, in splenic immune cells at single-cell resolution. A close relationship between B:T cell ratio and immunosenescence was detected, as B:T cell ratio was much higher in mouse than long-lived mammals and significantly increased during aging. Importantly, we identified several iron-related genes that could resist immunosenescence changes, especially the iron chaperon, PCBP1, which was upregulated in long-lived mammals but dramatically downregulated during aging in all splenic immune cell types. Supportively, immune cells of mouse spleens contained more free iron than those of bat spleens, suggesting higher level of ROS-induced damage in mouse. PCBP1 downregulation during aging was also detected in hepatic but not pulmonary immune cells, which is consistent with the crucial roles of spleen and liver in organismal iron recycling. Furthermore, PCBP1 perturbation in immune cell lines would result in cellular iron dyshomeostasis and senescence. Finally, we identified two transcription factors that could regulate PCBP1 during aging. Together, our findings highlight the importance of iron homeostasis in splenic anti-immunosenescence, and provide unique insight for improving human healthspan.

Spatially resolved expression landscape and gene-regulatory network of human gastric corpus epithelium.

Molecular knowledge of human gastric corpus epithelium remains incomplete. Here, by integrated analyses using single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, and single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) techniques, we uncovered the spatially resolved expression landscape and gene-regulatory network of human gastric corpus epithelium. Specifically, we identified a stem/progenitor cell population in the isthmus of human gastric corpus, where EGF and WNT signaling pathways were activated. Meanwhile, LGR4, but not LGR5, was responsible for the activation of WNT signaling pathway. Importantly, FABP5 and NME1 were identified and validated as crucial for both normal gastric stem/progenitor cells and gastric cancer cells. Finally, we explored the epigenetic regulation of critical genes for gastric corpus epithelium at chromatin state level, and identified several important cell-type-specific transcription factors. In summary, our work provides novel insights to systematically understand the cellular diversity and homeostasis of human gastric corpus epithelium in vivo.

Predictors of Comorbid Conditions in Women Who Carry an FMR1 Premutation.

Purpose: Women who carry an FMR1 premutation (PM) can experience two well-established PM-associated disorders: fragile X-associated primary ovarian insufficiency (FXPOI, affects ~20-30% carriers) and fragile X-associated tremor-ataxia syndrome (FXTAS, affects ~6-15% carriers); however, emerging evidence indicates that some of these women experience complex health profiles beyond FXPOI and FXTAS. Methods: In an effort to better understand predictors for these comorbid conditions, we collected self-reported medical histories on 413 women who carry an FMR1 PM. Results: There were 22 health conditions reported by at least 9% of women. In an exploratory analysis, 12 variables were tested in logistic regression models for each comorbid condition, including demographic variables, environmental variables, PM-associated factors, and endorsement of depression and/or anxiety. More than half of the comorbid conditions studied were associated with women who self-reported having anxiety. Age, smoking, body mass index (BMI), and depression were also significant predictor variables for specific comorbid conditions. Conclusions: Age, smoking, and BMI were significantly associated with a subset of the comorbid conditions analyzed. Importantly, depression or anxiety were also significantly associated with many of the comorbid health conditions. This work highlights some of the modifiable factors associated with complex health profiles among women with an FMR1 PM.

Refining the risk for fragile X-associated primary ovarian insufficiency (FXPOI) by FMR1 CGG repeat size.

PURPOSE: Approximately 20-30% of women with an FMR1 premutation experience fragile X-associated primary ovarian insufficiency (FXPOI); however, current risk estimates based on repeat size only identify women with the midrange of repeats to be at the highest risk. METHODS: To better understand the risk by repeat size, we collected self-reported reproductive histories on 1,668 women and divided them into high-resolution repeat size bins of ~5 CGG repeats to determine a more accurate risk for FXPOI in relation to CGG repeat length. RESULTS: As previously reported, women with 70-100 CGG repeats were at the highest risk for FXPOI using various statistical models to compare average age at menopause and risk of FXPOI, with women with 85-89 repeats being at the highest risk. Importantly, women with <65 repeats or >120 repeats did not have a significantly increased risk for FXPOI compared to women with <45 repeats. CONCLUSION: Using a large cross-section study on 1,668 women, we have provided more personalized risk assessment for FXPOI using high-resolution repeat size bins. Understanding the variability in risk has important implications for family planning and overall health among women with a premutation.

Study of telomere length in men who carry a fragile X premutation or full mutation allele.

The fragile X premutation is defined by the expansion of the CGG trinucleotide repeat at the 5' UTR of the FMR1 gene to between 55 and 200 repeats, while repeat tracks longer than 200 are defined as full mutations. Men carrying a premutation are at increased risk for fragile X-associated tremor/ataxia syndrome (FXTAS); those with > 200 repeats have fragile X syndrome, a common genetic form of intellectual disabilities. In our study, we tested the hypothesis that men carrying a fragile X premutation or full mutation are "biologically older", as suggested by the associated age-related disorder in the presence of the fragile X premutation or the altered cellular pathology that affects both the fragile X premutation and full mutation carriers. Thus, we predicted that both groups would have shorter telomeres than men carrying the normal size repeat allele. Using linear regression models, we found that, on average, premutation carriers had shorter telomeres compared with non-carriers (n = 69 vs n = 36; p = 0.02) and that there was no difference in telomere length between full mutation carriers and non-carriers (n = 37 vs n = 29; p > 0.10). Among premutation carriers only, we also asked whether telomere length was shorter among men with vs without symptoms of FXTAS (n = 28 vs n = 38 and n = 27 vs n = 41, depending on criteria) and found no evidence for a difference (p > 0.10). Previous studies have shown that the premutation is transcribed whereas the full mutation is not, and the expanded repeat track in FMR1 transcript is thought to lead to the risk for premutation-associated disorders. Thus, our data suggest that the observed premutation-only telomere shortening may be a consequence of the toxic effect of the premutation transcript and suggest that premutation carriers are "biologically older" than men carrying the normal size allele in the same age group.

Clustering of comorbid conditions among women who carry an FMR1 premutation.

PURPOSE: Emerging evidence indicates that women who carry an FMR1 premutation can experience complex health profiles beyond the two well-established premutation-associated disorders: fragile X-associated primary ovarian insufficiency (FXPOI, affects ~20-30% carriers) and fragile X-associated tremor-ataxia syndrome (FXTAS, affects ~6-15% carriers). METHODS: To better understand premutation-associated health profiles, we collected self-reported medical histories on 355 carrier women. RESULTS: Twenty-two health conditions were reported by at least 10% of women. Anxiety, depression, and headaches were reported by more than 30%. The number of comorbid conditions was significantly associated with body mass index (BMI) and history of smoking, but not age. Survival analysis indicated that women with FXPOI had an earlier age at onset for anxiety and osteoporosis than women without FXPOI. Cluster analysis identified eight clusters of women who reported similar patterns of comorbid conditions. The majority of carriers (63%) fell into three categories primarily defined by the presence of only a few conditions. Interestingly, a single cluster defined women with symptoms of FXTAS, and none of these women had FXPOI. CONCLUSION: Although some women with a premutation experience complex health outcomes, most carriers report only minimal comorbid conditions. Further, women with symptoms of FXTAS appear to be distinct from women with symptoms of FXPOI.

Differential expression of α6 and ß1 integrins reveals epidermal heterogeneity at single-cell resolution.

Skin epidermis is a constantly renewing epithelium that is composed of various cell types and provides an ideal model system for tissue regeneration and heterogeneity studies. Integrins are a family of transmembrane receptors that mediate cell adhesion in the epidermis, and integrin expression spatially reflects epidermal heterogeneity. It remains unclear whether differential expression of integrins can characterize cell types in skin epidermis. This study applied a fluorescence-activated cell sorting (FACS) strategy based on differential expression of α6 and ß1 integrins, and used transcriptome analysis to explore epidermal heterogeneity. First, epidermal cells were acquired from C57BL/6 mice back skin. Nine bulk-cell populations were sorted with differential expressions of α6 and ß1 integrins, and were successfully characterized as the main cell types in the epidermis through RNA-seq and transcriptome analysis. Then, tetOKrt14-H2BGFP mice were used to trace the cell proliferation rate during wound healing with GFP intensity. Epidermal cells were acquired from the re-epithelialized back skin wounds, and a total of 576 single cells were sorted, combining integrin expression and GFP tracing. FACS single-cell RNA-seq enabled high resolution in the classification of subtypes in both interfollicular epidermis and hair follicle, and both quiescent and intermediate cell states of the basal and infundibulum stem cell populations were distinguished. This study proposed a presorted method to investigate the relationship between integrin expression and epidermal heterogeneity. Multiple epidermal cell types and their expression profiles were identified, which provides data resources for dermatology research.

Dynamic expression of α6 integrin indicates epidermal cell behaviors.

Skin epidermis is a stratified epithelium that composed of interfollicular epidermis (IFE) and hair follicles (HFs). Integrins are cell-cell and cell-matrix adhesive ligands that play important roles in epidermal cell proliferation, migration and differentiation behaviors. Here, we analyzed the expression of both α6 and ß1 integrins. In vitro epidermal cell culture, both α6 and ß1 integrins displayed downregulation upon high Ca2+ induced differentiation. During wound healing (WH), α6 integrin showed dynamic expression, first greatly upregulated in unclosed wounds and then downregulated upon re-epithelialization. Further analysis of different wound regions confirmed α6 integrin significantly increased in migratory cells and migration was coupled with differentiation. However, expression level of ß1 integrin did not show significant correlation with migration. We discovered that α6 integrin directly indicates epidermal cell differentiation and wound directed migration behaviors with its expression level.

FXPOI: Pattern of AGG Interruptions Does not Show an Association With Age at Amenorrhea Among Women With a Premutation.

Fragile X-associated primary ovarian insufficiency (FXPOI) occurs in about 20% of women who carry a premutation allele (55-200 CGG repeats). These women develop hypergonadotropic hypogonadism and have secondary amenorrhea before age 40. A non-linear association with repeat size and risk for FXPOI has been seen in multiple studies women with a premutation: those with a mid-range of repeats are at highest risk (∼70-100 CGG repeats). Importantly, not all carriers with 70-100 repeats experience FXPOI. We investigated whether AGG interruptions, adjusted for repeat size, impacted age at secondary amenorrhea. We have reproductive history information and AGG interruption data on 262 premutation women: 164 had an established age at amenorrhea (AAA) (for some, age at onset of FXPOI) or menopause, 16 had a surgery involving the reproductive system such as a hysterectomy, and 82 women were still cycling at the last interview. Reproductive status was determined using self-report reproductive questionnaires and interviews with a reproductive endocrinologist. For each of these 262 women, FMR1 repeat size and number of AGG interruptions were determined. We confirmed the association of repeat size with AAA or menopause among women with a premutation. As expected, both premutation repeat size and the quadratic form of repeat size (i.e., squared term) were significant in a survival analysis model predicting AAA (p < 0.0001 for both variables). When number of AGG interruptions was added to the model, this variable was not significant (p = 0.59). Finally, we used a regression model based on the 164 women with established AAA to estimate the proportion of variance in AAA explained by repeat size and its squared term. Both terms were again highly significant (p < 0.0001 for both), but together only explained 13% of the variation in AAA. The non-linear association between AAA and FMR1 repeat size has been described in several studies. We have determined that AGG interruption pattern does not contribute to this association. Because only 13% of the variation is described using repeat size, it is clear that further research of FXPOI is needed to identify other factors that affect the risk for FXPOI.

Women who carry a fragile X premutation are biologically older than noncarriers as measured by telomere length.

Women who carry a fragile X premutation, defined as having 55-200 unmethylated CGG repeats in the 5' UTR of the X-linked FMR1 gene, have a 20-fold increased risk for primary ovarian insufficiency (FXPOI). We tested the hypothesis that women with a premutation + FXPOI have shorter telomeres than those without FXPOI because they are "biologically older." Using linear regression, we found that women carrying a premutation (n = 172) have shorter telomeres and hence, are "biologically older" than women carrying the normal size allele (n = 81). Strikingly, despite having shorter telomeres, age was not statistically associated with their telomere length, in contrast to non-carrier controls. Further, telomere length within premutation carriers was not associated with repeat length but was associated with a diagnosis of FXPOI, although the latter finding may depend on FXPOI age of onset.

Knockdown of Ras-Related Protein 25 (Rab25) Inhibits the In Vitro Cytotoxicity and In Vivo Antitumor Activity of Human Glioblastoma Multiforme Cells.

Ras-related protein 25 (Rab25) is a member of the Rab family, and it has been reported to play an important role in tumorigenesis. However, its direct involvement in human glioblastoma multiforme (GBM) is still unclear. The aim of the current study was to investigate the potential role of Rab25 in the growth, proliferation, invasion, and migration of human GBM. Our results showed that Rab25 expression was significantly higher in human GBM cell lines compared with a normal astrocyte cell line. In vitro functional studies revealed that knockdown of Rab25 reduced cell proliferation and inhibited invasion and migration of GBM cells. In vivo experiments showed that knockdown of Rab25 attenuated the tumor growth in nude mice. Finally, knockdown of Rab25 significantly inhibited the phosphorylation levels of PI3K and AKT in GBM cells. Taken together, these findings indicate that Rab25 may act as a tumor promoter in human GBM and that approaches to target Rab25 may provide a novel strategy to treat this disease.

Neuroprotective mechanism of HIF-1α overexpression in the early stage of acute cerebral infarction in rats.

The present study aimed to explore the expression and neuroprotective mechanism of hypoxia inducible factor (HIF-1α) in the brain tissue of a rat model of early acute cerebral infarction. A total of 64 Sprague Dawley rats were randomly divided into surgery and sham groups and the model of focal cerebral infarction was established by the suture-occluded method. In the sham group, blood vessels were separated but not occluded. Rats in the surgery and sham groups were subdivided into eight groups (n=4/group). Blood samples was collected at 8 time points including 30 min and 1, 3, 6, 12, 48, 24 and 72 h, respectively, and HIF-1α content was detected using ELISA. Brain tissues of rats in all groups were harvested following blood collection. HIF-1α protein expression was detected by immunohistochemistry and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling was used to analyze the brain cell apoptosis index. ELISA results demonstrated that rats in the surgery group began to express HIF-1α within 30 min, and HIF-1α expression levels gradually increased, peaking at 12 h. HIF-1α expression levels were significantly increased in the surgery group at all time points, as compared with the sham group (P<0.05). The concentration of HIF-1α decreased rapidly in 12 h. At various time points, HIF-1α protein expression in the brain tissue of rats in the sham group was negative. HIF-1α protein expression was significantly increased in the surgery group (P<0.05), peaking at 12 h, and decreasing after this point. As compared with the sham group, the apoptosis indices of the brain tissue of rats in the surgery group exhibited a gradual increasing trend with significant decreases observed after 12 h (P<0.05). Intra-group comparison of all indices in the surgery group, indicated that there was a statistically significant difference between postoperative 12 h and other time points (P<0.05). In conclusion, the present study demonstrated that HIF-1α was highly expressed in the brain tissue of rat models of early acute cerebral infarction. The results also indicated that HIF-1α significantly reduced the apoptosis of infarcted cells, suggesting that HIF-1α may have a neuroprotective role in early acute cerebral infarction.

Expression of TYMS in lymph node metastasis from low-grade glioma.

The aim of the present study was to investigate the expression of thymidylate synthase (TYMS) in the primary foci and metastatic lymph nodes of low-grade glioma, and to analyze the function of TYMS in the lymph node metastases from low-grade glioma. The study included 93 cases of surgically resected and pathologically confirmed low-grade glioma, form patients treated at Huaihe Hospital of Henan University (Kaifeng, China). The following clinical data was obtained from each patient: Gender, age, subjective symptoms (dizziness, headache, a feeling of pressure in the head, etc.), site of disease, tumor type, pathological stage, degree of differentiation and lymph node involvement. The surgically resected gliomas and dissected cervical lymph nodes were immunohistochemically stained, and DNA was extracted from the tumor and lymph node tissues samples for polymerase chain reaction sequencing and amplification. The expression of TYMS in the primary foci and metastatic lymph nodes of low-grade glioma was examined. Additionally, the association between pathological features and the postoperative survival rate of the patients was analyzed. The primary lesions of all 93 cases exhibited positive TYMS expression and 43/157 (27.39%) lymph nodes exhibited positive TYMS expression. Factors that significantly influenced the postoperative survival rate of the patients, included the metastasis of the cervical lymph nodes (P<0.01), the number of dissected cervical lymph nodes (P<0.01) and the degree of differentiation (P<0.05). The metastasis of the cervical lymph nodes was the only independent risk factor affecting postoperative disease-free survival. The risk of recurrence in patients with metastasis of the cervical lymph nodes was 6.3-fold higher than in those without metastasis (P<0.01). Thus, the results of the present study provide a theoretical basis for accurately predicting the prognosis of patients with low-grade malignant brain glioma, reducing the conjecture involved in selecting postoperative treatment strategies and improving therapeutic efficacy.

Prenatal alcohol exposure inducing the apoptosis of mossy cells in hippocampus of SMS2-/- mice.

In order to understand the mechanisms of alcohol-induced neuroapoptosis through the ceramide pathway, sphingomyelin synthase 2 knockout (SMS2-/-) mice were used to make the prenatal alcohol exposure model, and the role of ceramide regulation on alcohol-induced neuroapoptosis was studied in the offspring. Initially the levels of serum sphingomyelin (SM) were detected with enzymatic method in P0 pups after alcohol exposure in parents. Then the apoptosis of mossy cells in the offspring hippocampus was investigated after prenatal alcohol exposure with immunohistochemistry and TUNEL assay. Finally the expression of activated Caspase 8 and activated Caspase 3 in the offspring hippocampus was detected with Western blot analysis. Our results showed that SM levels were down-regulated in a dose-dependent manner (p<0.05) after prenatal alcohol exposure in wild-type (WT) and SMS2-/- pups. However, SM levels of serum in SMS2-/- pups were significantly lower than that in WT pups (p<0.01). Furthermore, we found that mossy cells were very sensitive to alcohol-induced neuroapoptosis. In both WT pups and SMS2-/- pups, the number of apoptotic mossy cells in the hippocampus increased after prenatal alcohol exposure in a dose dependent manner (p<0.05) and decreased with the growing age. Compared with WT pups, the number of apoptotic mossy cells in the hippocampus of SMS2-/- pups increased (p<0.05). Western blotting showed that the expression of activated Caspase 8 and activated Caspase 3 of hippocampal tissue in WT pups and SMS2-/- pups increases after prenatal alcohol exposure, consistent with results from TUNEL assay and immunocytochemistry. Our study suggests that mossy cells may be the easily attacked cells for fetal alcohol spectrum disorder (FASD), and ceramide is involved in the alcohol-induced neural apoptosis. The mechanism probably lies in the accumulated ceramide in SMS2 mice, and the increase of activated Caspase 8 and Caspase 3 promotes alcohol-induced neuroapoptosis.

The role of arginine vasopressin in electroacupuncture treatment of primary sciatica in human.

It has been implicated that electroacupuncture can relieve the symptoms of sciatica with the increase of pain threshold in human, and arginine vasopressin (AVP) in the brain rather than the spinal cord and blood circulation participates in antinociception. Our previous study has proven that AVP in the brain played a role in the process of electroacupuncture analgesia in rat. The goal of the present study was to investigate the role of AVP in electroacupuncture in treating primary sciatica in human. The results showed that (1) AVP concentration of cerebrospinal fluid (CSF) (7.5 ± 2.5 pg/ml), not plasma (13.2 ± 4.2 pg/ml) in primary sciatica patients was lower than that in health volunteers (16.1 ± 3.8 pg/ml and 12.3 ± 3.4 pg/ml), although the osmotic pressure in CSF and plasma did not change; (2) electroacupuncture of the bilateral "Zusanli" points (St. 36) for 60 min relieved the pain sensation in primary sciatica patients; (3) electroacupuncture increased the AVP level of CSF, not plasma in primary sciatica patients; and (4) there was the positive correlation between the effect of electroacupuncture relieving the pain and the AVP level of CSF in the primary sciatica patients. The data suggested that central AVP, not peripheral AVP might improve the effect of electroacupuncture treatment of primary sciatica in human, i.e., central AVP might take part in the electroacupuncture relieving the pain sensation in primary sciatica patients.

Gene expression profiles of entorhinal cortex in Alzheimer's disease.

The incidence of Alzheimer's disease (AD) has been increasing in the recent years but the underlying mechanisms remain uncertain. This study aimed to analyze the differentially expressed genes (DEGs) in entorhinal cortex with AD and identify featured genes related to AD. Gene expression profile GSE5281 was downloaded from Gene Expression Omnibus, including 10 AD and 13 control samples. Differentially expressed genes were identified by Student t test including 119 upregulated and 591 downregulated DEGs. Then, we obtained 14 enrichment Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways among which 11 pathways were significantly enriched (adjusted P value < .05). The KEGG pathway network which was constructed by 14 KEGG pathways showed that 6-phosphofructokinase, muscle type, phosphoglucomutase 1, aldolase A, and adolase C had high degree. Glycometabolism pathways network which was constructed by 4 glycometabolism pathways showed that adenosine triphosphate (ATP) synthase, H+transporting, mitochondrial F1 complex ATP5B, ATP5C1, ATP5D, and ATP5G1 had high degree related to ATP metabolism. These findings suggested that these genes with high degree may be the underlying potential therapeutic targets for AD.

Examination of FMR1 transcript and protein levels among 74 premutation carriers.

Fragile X-associated disorders are caused by a CGG trinucleotide repeat expansion in the 5'-untranslated region of the FMR1 gene. Expansion of the CGG trinucleotide repeats to >200 copies (that is, a full mutation) induces methylation of the FMR1 gene, with transcriptional silencing being the eventual outcome. Previous data have shown that FMR1 premutation carriers (individuals with 55-199 repeats) have increased FMR1 mRNA levels with decreased protein (fragile X mental retardation protein (FMRP)) levels. However, the point at which this translational inefficiency occurs, given the increased transcription mechanism, has not yet been explored and remains to be elucidated. We examined the repeat length group, FMR1 transcript and FMRP levels in 74 males with a wide range of repeat lengths using analysis of covariance to better characterize this association. Results showed that the mean FMRP level among carriers with 80-89 repeats was significantly higher than the mean levels among lower (54-79) and higher (90-120) premutation carriers, in spite of the increasing transcript level with repeat length. Taken together, these results suggest that the 80-89-repeat group may lead to different properties that increase the efficiency of translation compared with other premutation repeat size groups.

A study of the distributional characteristics of FMR1 transcript levels in 238 individuals.

Fragile X syndrome, the most common form of inherited mental retardation, is caused by hyperexpansion and hypermethylation of a CGG repeat tract in the 5' untranslated region of the FMR1 gene. This methylation causes the gene to be transcriptionally silenced. In addition to the common allele form with less than 41 repeats, there are two other allelic forms of the FMR1 gene that are unmethylated: premutation (61-200 CGG repeats) and intermediate (41-60 CGG repeats). Recently, premutation-specific phenotypes not related to fragile X syndrome have been reported: a 20-fold increased risk for premature ovarian failure (POF) among female carriers and an increased risk for a tremor ataxia syndrome (TAS) primarily among older male carriers. At the molecular level, increased levels of FMR1 transcript have been observed among premutation carriers. Increased levels of transcript may be causally related to the POF or TAS phenotypes or may be a surrogate of some other allelic property. In this report, we have examined the distributional properties of transcript levels by repeat size and gender among 238 individuals. We have confirmed a significant linear relationship between transcript level and repeat size in males and females. The evidence for the linear effect is primarily within the premutation size alleles.

Sequence analysis of the mouse IRBP gene and cDNA.

PURPOSE: To understand the structure of the mouse interphotoreceptor retinoid-binding protein (IRBP) gene and to compare the predicted primary structure within each repeat of IRBP with its relatives. To compare the levels of expression of IRBP RNA in normal and knockout mice. METHODS: The DNA sequence was determined by sub-cloning restriction fragments of the IRBP gene from 129/Sv P1 clones. Primers were designed to utilize the walking approach. Additional sequences were obtained by PCR amplification from genomic DNA and direct sequencing of products. Mouse retina RNA was subjected to reverse transcription coupled to PCR and the accumulation of double stranded DNA product was monitored with SYBR Green. The PCR primers flanked Intron C, to avoid the analysis of contaminating genomic DNA. RESULTS: Altogether a contig was assembled with a final length of about 14.4 kb. The mouse gene structure is similar to the pattern of exons and introns in the bovine and human genes, with a long first exon encoding most of the protein. The splice site boundaries closely match consensus sequences and the exons appear to be identically placed among the three species (bovine, human, and mouse). A region containing a repeated sequence of low complexity is located about 1.75 to 1.4 kb upstream of the transcription start site. A second region containing another low complexity repeat is found in Intron C close to the end of Exon 3. A limited number of weak consensus polyadenylation signals in the 3' region suggest at least three different transcription terminators that apparently give rise to the previously known mouse IRBP mRNAs. The mRNA for IRBP was detected in normal and part of the mRNA was detected in the IRBP knockout mouse, consistent with previous observations. The level of the IRBP mRNA remnant was reduced about 10 fold in the knockout mouse, also consistent with the previously reported absence of Repeat 4 immunoreactivity. CONCLUSIONS: The strong conservation in intron-exon positions, gene structure, and protein sequence among mammals supports an important biological role for these signals and for the IRBP protein in vision. Low levels of aberrant IRBP mRNA in the knockout mouse are consistent with no immunologically detectable Repeat 4 protein in this mouse.