Distribution of class I, III and IV alcohol dehydrogenase mRNAs in the adult rat, mouse and human brain.

The localization of different classes of alcohol dehydrogenases (ADH) in the brain is of great interest because of their role in both ethanol and retinoic acid metabolism. Conflicting data have been reported in the literature. By Northern blot and enzyme activity analyses only class III ADH has been detected in adult brain specimens, while results from riboprobe in situ hybridization indicate class I as well as class IV ADH expression in different regions of the rat brain. Here we have studied the expression patterns of three ADH classes in adult rat, mouse and human tissues using radioactive oligonucleotide in situ hybridization. Specificity of probes was tested on liver and stomach control tissue, as well as tissue from class IV ADH knock-out mice. Only class III ADH mRNA was found to be expressed in brain tissue of all three investigated species. Particularly high expression levels were found in neurons of the red nucleus in human tissue, while cortical neurons, pyramidal and granule cells of the hippocampus and dopamine neurons of substantia nigra showed moderate expression levels. Purkinje cells of cerebellum were positive for class III ADH mRNA in all species investigated, whereas granular layer neurons were positive only in rodents. The choroid plexus was highly positive for class III ADH, while no specific signal for class I or class IV ADH was detected. Our results thus support the notion that the only ADH expressed in adult mouse, rat and human brain is class III ADH.     

Molecular cloning of porcine alpha 1-microglobulin/HI-30 reveals developmental and tissue-specific expression of two variant messenger ribonucleic acids.

A 1008 basepair (bp) cDNA clone encoding 335 amino acids followed by an inframe TGA translation termination codon and a 295-nucleotide 3' untranslated (UT) region has been isolated from a pig liver cDNA library. Based on the deduced amino acid and nucleotide sequence homology to a human cDNA (Kaumeyer, J.F., Polazzi, J.O. and Kotick, M.P. (1986) Nucleic Acids Res. 14, 7839-7850), the 5' amino terminus was found to code for alpha 1-microglobulin (alpha 1-M), a 183 amino acid protein belonging to the lipocalin protein superfamily (Pervaiz, S. and Brew, K. (1985) Science 228, 335-337). The 3' half encoded HI-30 which constitutes the Kunitz-type proteinase inhibitory (L-chain) domain of porcine inter-alpha-trypsin inhibitor (I alpha TI). In Northern blot hybridization, this cDNA identified two equally abundant mRNA species of approx. 1.3 kb and 1.6 kb in length. However, a 125 bp cDNA probe derived from the 3' UT region of the cDNA hybridized only to the 1.6 kb mRNA. The differences observed in the 3' UT region of these mRNAs suggest the utilization of alternative polyadenylation signals or presence of unprocessed nuclear RNA. Densitometric scanning of Northern blots indicated that alpha 1-M/HI-30 mRNA levels were higher (5-8-fold) in fetal and neonatal liver compared to that of primiparous pigs. In contrast, the RNA levels did not change significantly during pregnancy. Dot blot analysis of RNA indicated liver to be the major site of alpha 1-M/HI-30 mRNA expression with lower levels observed in the stomach. The results suggest that modulation of alpha 1-M/HI-30 gene expression could play a role during porcine growth. Increased I alpha TI L-chain mRNA levels may be particularly important in fetal and neonatal development when regulation of the inflammatory response and protection of macromolecules from proteolytic degradation is vital to survival and sustained growth.     

Regulation of rat liver maturation in vitro by glucocorticoids.

The biochemistry of liver maturation was studied by using the RLA209-15 fetal rat hepatocyte line that is temperature sensitive for maintenance of the differentiated fetal liver phenotype. At 33 degrees C these cells were dedifferentiated; but at 40 degrees C they were phenotypically differentiated and, like normal fetal hepatocytes, synthesized moderate levels of albumin and transferrin, high levels of authentic (69,000 and 73,000 molecular weight) rat fetal alpha-fetoprotein (AFP), and low levels of a 65,000-molecular-weight variant AFP. Our results indicated that administration of glucocorticoid hormones to RLA209-15 cells at 40 degrees C induced a series of events associated with normal hepatocyte maturation; synthesis of fetal AFP was inhibited, whereas the synthesis of variant AFP, albumin, transferrin, tyrosine aminotransferase, and alpha 1-acid glycoprotein was induced. The variant AFP was produced by RLA209-15 cells at both temperatures and was encoded by an mRNA of 1.7 kilobases (kb). The fetal AFP was encoded by an mRNA of 2.2 kb. Normal adult rat liver contained three AFP mRNAs of 2.2 (minor), 1.7, and 1.5 kb. The 1.7-kb adult liver AFP mRNA comigrated with the RNA found in RLA209-15 cells, and both directed the synthesis of a 50,000-molecular-weight precursor polypeptide of the variant AFP. Administration of glucocorticoids to RLA209-15 cells grown at 33 degrees C stimulated synthesis of both the fetal and variant AFPs, but the levels of the 2.2-kb AFP mRNA were preferentially increased. RLA209-15 cells contained two glucocorticoid receptor mRNAs of 6.8 and 4.5 kb. The glucocorticoid-mediated maturation described above was blocked by the antiglucocorticoid RU486.     

Identification of a cDNA clone that contains the complete coding sequence for a 140-kD rat NCAM polypeptide

Neural cell adhesion molecules (NCAMs) are cell surface glycoproteins that appear to mediate cell-cell adhesion. In vertebrates NCAMs exist in at least three different polypeptide forms of apparent molecular masses 180, 140, and 120 kD. The 180- and 140-kD forms span the plasma membrane whereas the 120-kD form lacks a transmembrane region. In this study, we report the isolation of NCAM clones from an adult rat brain cDNA library. Sequence analysis indicated that the longest isolate, pR18, contains a 2,574 nucleotide open reading frame flanked by 208 bases of 5' and 409 bases of 3' untranslated sequence. The predicted polypeptide encoded by clone pR18 contains a single membrane-spanning region and a small cytoplasmic domain (120 amino acids), suggesting that it codes for a full-length 140-kD NCAM form. In Northern analysis, probes derived from 5' sequences of pR18, which presumably code for extracellular portions of the molecule hybridized to five discrete mRNA size classes (7.4, 6.7, 5.2, 4.3, and 2.9 kb) in adult rat brain but not to liver or muscle RNA. However, the 5.2- and 2.9-kb mRNA size classes did not hybridize to either a large restriction fragment or three oligonucleotides derived from the putative transmembrane coding region and regions that lie 3' to it. The 3' probes did hybridize to the 7.4-, 6.7-, and 4.3-kb message size classes. These combined results indicate that clone pR18 is derived from either the 7.4-, 6.7-, or 4.3-kb adult rat brain RNA size class. Comparison with chicken and mouse NCAM cDNA sequences suggests that pR18 represents the amino acid coding region of the 6.7- or 4.3-kb mRNA. The isolation of pR18, the first cDNA that contains the complete coding sequence of an NCAM polypeptide, unambiguously demonstrates the predicted linear amino acid sequence of this probable rat 140-kD polypeptide. This cDNA also contains a 30-base pair segment not found in NCAM cDNAs isolated from other species. The significance of this segment and other structural features of the 140-kD form of NCAM can now be studied.     

Species specificity and developmental patterns of expression of the beta amyloid precursor protein (APP) gene in brain, liver and choroid plexus in birds.

1. Human APP cDNA hybridized to a 3.5 kb mRNA in liver and brain RNA from chickens, pigeons, quail and ducks as well as in RNA from choroid plexus of chicken and quail. In contrast to all other species hitherto examined a 1.6 kb mRNA hybridizing to APP cDNA was found in abundant amounts in RNA from chicken and quail livers. 2. In the chicken, before hatching, the levels of APP mRNA in total RNA from liver and choroid plexus were higher than those in RNA from liver and choroid plexus of adults. However, RNA from the rest of the brain of chicken embryos contained less APP mRNA than RNA from brain of adults. 3. In the chicken, between 10 and 40 days after hatching, APP mRNA levels in RNA from liver were higher than adult levels, APP mRNA levels in RNA from choroid plexus were similar to adult levels and APP mRNA levels in RNA from the rest of brain were below the adult levels.     

Multiple polyadenylation sites in a Drosophila tropomyosin gene are used to generate functional mRNAs.

The gene encoding muscle tropomyosin I in Drosophila is alternatively spliced in embryonic and thoracic muscle to generate two sizes classes of RNAs. By Northern blot analysis, the embryonic RNA class shows a broad RNA band of hybridization of 1.3 kb and a more sharply defined, less abundant RNA band at 1.6 kb. The thoracic class of RNAs, on the other hand, consists of a broad hybridization band at 1.7 kb and a more sharply defined band at 1.9 kb. Each size class of RNA encodes a different tropomyosin isoform. The two classes of alternatively spliced RNAs utilize the same 3' terminal exon of the gene. The DNA sequence of this exon reveals a cluster of several polyadenylation signals (AAUAAA) or polyadenylation-like signals. We show here by S1 nuclease protection analysis that at least five and possibly seven of these polyadenylation or polyadenylation-like sequences are associated with in vivo embryonic and thoracic mRNA cleavage processing sites. Six of these S1 sites are clustered within 119 bp and a seventh is located 255 bp downstream. At least one of the polyadenylation-like signal sequences appears to be an unusual AACAAA sequence. In addition we also show that these mRNAs function in vitro to synthesize muscle tropomyosins.     

Tissue specific expression of rat peptidylglycine alpha-amidating monooxygenase activity and mRNA

The tissue specific expression of peptidylglycine alpha-amidating monooxygenase [(PAM) EC 1.14.17.3], an enzyme which catalyzes the formation of amidated bioactive peptides from their glycine-extended precursors, was examined in adult rat. Soluble and membrane-associated PAM enzymatic activities were determined, and the levels and size classes of PAM mRNA were examined by Northern blot analysis. PAM specific activity varied 1000-fold in the tissues examined, with highest levels in heart atrium, pituitary and salivary glands, and hypothalamus. The fraction of total PAM activity that was membrane associated varied from approximately 70% in heart atrium to 10% in neurointermediate pituitary lobe and thyroid gland. Levels of PAM mRNA varied over 300-fold. In the heart atrium, PAM mRNA accounts for more than 0.1% of the mRNA. For many tissues the ratio of total PAM specific activity to PAM mRNA levels was similar; however, PAM activity was higher than expected from mRNA levels in the salivary glands and lower than expected in several tissues, including heart ventricle. Three major size classes of PAM mRNA were identified among the tissues. Use of RNAse H indicated that differences in size were not due to the length of the poly(A) tail. The heart and central nervous system expressed PAM mRNA of the 4.2 kilobase (kb) and 3.8 kb size classes, while the remaining tissues expressed predominantly 3.8 kb and 3.6 kb classes; few tissues contained only one size class of PAM mRNA. The two major forms of PAM mRNA in adult heart atrium differ by the presence or absence of a 315 nucleotide segment in the protein coding region. Using a cDNA probe from within this segment, the 4.2 kb and 3.8 kb size classes of PAM mRNA in the central nervous system appeared to resemble those in the heart atrium. In the remaining tissues, a subset of PAM mRNAs in the 3.8 kb and 3.6 kb size classes hybridized with this probe, suggesting that additional forms of PAM mRNA are present.     

Structure and expression of the human haptoglobin locus.

Human genomic clones of the haptoglobin Hp1F and the "haptoglobin related' gene (Hpr) have been isolated. The two genes are adjacent, spanning a region of approximately 21 kb. A comparison of their coding sequences shows that Hpr differs from Hp1F at 28 codons. Northern blot and primer elongation analyses with human liver RNA show that the haptoglobin gene Hp1F appears to be transcribed some 1000-fold less in fetal than in adult liver. In adult liver the amount of Hpr mRNA is at the lower limit of detection, therefore the extent of its expression is at most less than 1000-fold that of the Hp1F gene. No Hpr mRNA can be detected in fetal liver.