A deletion including the brain promoter of the Duchenne muscular dystrophy gene is not associated with mental retardation.

A total of 161 unrelated Duchenne (DMD) and Becker muscular dystrophy (BMD) patients were screened for deletions in the brain promoter region of the dystrophin gene. Southern blot analysis using a probe for the brain promoter detected a deletion in this region in only one of the DMD families, in a patient with normal intelligence. This deletion also included the promoter of the muscle-type dystrophin and the exons encoding the actin-binding and part of the spectrin-like domains. Our data suggest that deletions in the brain promoter region are rare in DMD and are compatible with normal intelligence.

Multiple products of the Duchenne muscular dystrophy gene.

The gene which is defective in Duchenne Muscular Dystrophy (DMD) extends over 2300 kb of the X chromosome. Its product in the muscle is a 14 kb mRNA encoding a 427 kd rod-shaped protein called dystrophin. A 14 kb transcript encoding a very similar isoform of dystrophin is produced in the brain. The brain 14 kb mRNA is transcribed from the same gene but controlled by a different promoter, located at least 75 kb upstream from the muscle dystrophin promoter. The regulation of these promoters is very stringently controlled. The muscle-type but not the brain-type dystrophin mRNA is found in cloned skeletal muscle cells and its presence is correlated with the appearance of multinucleated fibers. The brain type is expressed in neurons, while in glia cells the muscle-type promoter is active. A third DMD gene transcript which is only 6.5 kb long has been identified. It contains the sequence coding for the C-terminal domain and the cysteine-rich domain of dystrophin but not the large region encoding the spectrin-like repeats and the N-terminal domain. The cell type distribution of this transcript is also very different from that of the two 14 kb mRNA isoforms. It is the major product of the DMD gene in many nonmuscle tissues including brain. Using monoclonal antibodies we have identified a 77 Kd protein which seems to be the translation product of this mRNA. As expected from the distribution of the 6.5 Kb mRNA, this protein is the major DMD gene product detectable in brain and many other nonmuscle tissues; it is undetectable in skeletal muscle but is present in the heart and stomach (as is the 6.5 Kb mRNA).

Mapping of dystrophin brain promoter: a deletion of this region is compatible with normal intellect.

Using a mouse genomic fragment containing the brain-specific promoter region of the dystrophin gene, we have located the brain promoter 75-300 kb proximal of the muscle promoter. Within our DMD-families we detected a patient who lacks both the brain-specific and muscle-specific promoter sequences. The normal intellectual capabilities of the patient argue against an indispensable role of the brain-specific first exon in mental functioning. The possibility exists that a NH2-terminally truncated dystrophin has taken over the function of the normal dystrophins in brain and/or muscle.

Brain-type and muscle-type promoters of the dystrophin gene differ greatly in structure.

The promoter of the 14 kb mRNA encoding the brain isoform of dystrophin in the mouse has been isolated and partially characterized. Unlike the promoter of the muscle dystrophin isoform, it does not contain a TATA box or other consensus sequences characteristic of the proximal region upstream of the cap sites of eukaryotic genes. Yet, it has a major initiation of transcription start site located 266 bp upstream from the first ATG which is in frame with the dystrophin coding sequence. The 5' untranslated region contains nine additional ATG triplets which are not in-frame with the coding sequence or are followed by stop codons. A DNA fragment extending from bp -1149 to +11 is sufficient to activate a reporter gene lacking a promoter in transfected neuroblastoma cells.

An in situ-hybridization study of the localization of retinol-binding protein and transthyretin messenger RNAs during fetal development in the rat.

The patterns, and the cellular sites, of expression of the genes for retinol-binding protein (RBP) and transthyretin (TTR) were studied during early- to mid-stages of rat embryogenesis. In situ hybridization of single-stranded RNA probes to rat embryo sections revealed specific sites at which RBP messenger RNA (mRNA) and TTR mRNA were localized in rat conceptuses from 7 to 13 days of gestation. RBP and TTR mRNAs were both observed in the visceral endoderm as early as at 7 days of gestation. The transcripts were not expressed in the parietal endoderm. At 9 days of gestation, TTR mRNA was strongly detected in the visceral extraembryonic endoderm and in the foregut endoderm, whereas RBP mRNA was detected only in the visceral extraembryonic endoderm. From the 10th day to the 13th day of gestation, both transcripts were increasingly expressed in the visceral yolk sac endoderm and in the developing fetal liver, and gradually decreased in the foregut. At 11 days of gestation, TTR mRNA was first detected in the tela choroidea of the fourth ventricle in the brain; and at 13 days, the TTR mRNA was strongly evident in the developing choroid plexus of the fourth and lateral ventricles. These in situ hybridization studies with embryos at different developmental stages show that RBP and TTR mRNAs are transcribed quite early during embryogenesis. The protein products of these transcripts may play important roles in vitamin A and thyroid hormone metabolism, and in the functions that these regulatory compounds play, in the developing rat embryo.

Localization of retinol-binding protein messenger RNA in the rat kidney and in perinephric fat tissue.

The cellular localization of retinol-binding protein (RBP) messenger RNA (mRNA) in the kidney, and the developmental pattern of the renal expression of the RBP gene, were studied in the Sprague-Dawley rat. In situ hybridization studies were conducted with single-stranded cRNA probes, using sections of adult and young rat kidneys. These studies revealed specific localization of RBP mRNA in the outer stripe of the medulla, specifically localized in the S3 segment of the proximal tubules. Northern blot analysis demonstrated that RBP mRNA was not detectable in the kidney before birth or during the first week postpartum, but was clearly detected by the end of the second week of age. No RBP mRNA was observed in the kidney by in situ hybridization at 12 days of age. At 26 days of age, however, RBP mRNA was clearly detected by the in situ hybridization technique, localized in the same anatomic region as that observed in the adult kidney. Transthyretin mRNA was not detected in the adult kidney. Previous studies have shown that immunoreactive RBP is localized in the convoluted segment of the proximal tubules of the rat kidney. The present results demonstrate that RBP mRNA in the kidney is localized in an anatomic region (the S3 segment of the proximal tubules) different from that of immunoreactive RBP. In addition, an intense RBP mRNA hybridization signal was detected in the perinephric fat tissue of 26- and 40-day-old and adult rats. Further analysis of RNA from epididymal fat showed a level of RBP mRNA approximately 20% of that of liver. The function of RBP synthesized in the kidney and adipose tissue remains to be determined. We have previously hypothesized that RBP synthesized in extrahepatic tissue may function in the recycling of retinol back to the liver or to other target tissues.

Plasma transthyretin. Tissue sites of degradation and turnover in the rat.

Transthyretin (TTR) is involved in the plasma transport of both retinol and thyroid hormones. TTR is synthesized in the liver and choroid plexus, and in small amounts in several other tissues. A study was conducted to determine the tissue sites of degradation and turnover of TTR in the rat. The study employed TTR labeled with tyramine cellobiose (TC) and the trapped ligand method. Samples of purified rat TTR were labeled either with 125I-TC or directly with 131I. A mixture of the two labeled TTRs was injected intravenously into six rats. Blood samples were collected via a venous catheter for kinetic (turnover) analysis. After 24 or 48 h, the rats were killed, and 23 different tissues/organs were assayed as possible sites of TTR degradation. Derivatization of TTR with TC did not appreciably alter TTR plasma kinetics. Plasma turnover data were best fit by a three-pool model. The mean fractional turnover of plasma TTR was 0.15/h, and of total body TTR 0.04/h. The major sites of TTR degradation were the liver (36-38% of total body TTR degradation, almost all in hepatocytes), muscle (12-15%), and skin (8-10%). Tissues that were sites of 1-8% of body TTR degradation included kidneys, adipose tissue, testes, and the gastrointestinal tract. Less than 1% of total TTR degradation occurred in the other tissues examined. A second study was conducted in which labeled TTR was injected intraventricularly into the cerebrospinal fluid in order to explore the degradation of TTR of choroid plexus origin. The kinetics of the appearance and disappearance of such labeled TTR in plasma were physiologically reasonable, with an estimated turnover of cerebrospinal fluid TTR of the order of 0.33/h. The major tissue sites of degradation of labeled TTR injected into cerebrospinal fluid and into plasma were approximately the same. No specific degradation of TTR was found in the nervous system tissues. The most active organs of TTR catabolism, per gram wet weight, were liver and kidneys. These studies demonstrate that many tissues participate in TTR turnover and degradation; the studies provide quantitative information about the tissue sites of TTR catabolism.

Transthyretin: a choroid plexus-specific transport protein in human brain. The 1986 S. Weir Mitchell award.

Plasma transthyretin (TTR, formerly called prealbumin) is a 55-kd protein that participates in the plasma transport of both thyroxine and retinol (vitamin A). TTR concentrations are disproportionately high in human ventricular CSF, suggesting that TTR is either selectively transported across or synthesized de novo within the blood-CSF barrier. To address this question, we adopted a molecular genetic approach; after isolating a cDNA clone encoding human TTR, we previously demonstrated specific TTR messenger RNA (mRNA) synthesis in rat choroid plexus. We have now extended these investigations to the human brain. Northern analysis of postmortem brain homogenates revealed abundant TTR mRNA in choroid plexus, but not in cerebellum or cerebral cortex. Choroid plexus mRNA was readily translated into TTR preprotein in an in vitro translation system. An immunocytochemical survey of human postmortem brain sections revealed the presence of TTR protein specifically and uniquely in the cytoplasm of choroid plexus epithelial cells; these results were corroborated at the mRNA level by an extensive survey of whole rat-brain sections by in situ hybridization. Therefore, within the mammalian CNS, TTR is the first known protein synthesized solely by the choroid plexus, suggesting a special role for TTR in the brain or CSF. Whether this function differs from its established plasma transport functions is presently unknown.

Localization of immunoreactive transthyretin (prealbumin) and of transthyretin mRNA in fetal and adult rat brain.

We used a combination of immunohistochemical and molecular-biological techniques to investigate the localization of transthyretin (TTR) in the brains of adult and fetal rats. The immunohistochemical studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of TTR using the unlabeled peroxidase-antiperoxidase method. TTR mRNA levels were measured by Northern-blot analysis of poly (A+) RNA, followed by hybridization to 32P-labeled TTR cDNA; TTR mRNA was localized in brain tissue sections by in situ hybridization. Immunoreactive TTR was found to be specifically localized in the choroid plexus epithelial cells of adult rat brain. High levels of TTR mRNA were found in poly (A+) RNA samples obtained from the choroid plexus. In addition, the specific localization of TTR mRNA in the epithelial cells of the choroid plexus was demonstrated by in situ hybridization. Neither immunoreactive TTR nor TTR mRNA were found in other regions of adult rat brains. The levels of TTR mRNA in the choroid plexus were at least 30 times higher than those observed in the adult liver. Immunoreactive TTR was observed in the brains of fetal rats on as early as the 11th day of gestation. This immunoreactive TTR was localized in the tela choroidea, the developmental forerunner of the choroid plexus. Immunoreactive TTR was also observed in the fetal choroid plexus as it began to form (14th day of gestation) as well as in the more completely developed choroid plexus (18th day of gestation).(ABSTRACT TRUNCATED AT 250 WORDS)

Photo-affinity label for cellular retinol-binding protein.

A number of retinoid derivatives have been synthesized for use as labels for cellular retinol-binding protein. Introduction of substituents abolished the binding of the derivatives to the protein, except in the case of the photo-reactive derivative, 4-azidoretinol. This compound was found to compete successfully with all-trans-retinol for binding to cellular retinol-binding protein, with a high relative binding affinity. Irradiation of a complex of 4-azidoretinol and a semi-purified preparation of cellular retinol-binding protein from liver resulted in a firm attachment stable to SDS-gel electrophoresis. It is therefore suggested that the irradiated product is held together covalently. A method for the synthesis of 4-azidoretinol is described.