An iron-regulated ferric reductase associated with the absorption of dietary iron

The ability of intestinal mucosa to absorb dietary ferric iron is attributed to the presence of a brush-border membrane reductase activity that displays adaptive responses to iron status. We have isolated a complementary DNA, Dcytb (for duodenal cytochrome b), which encoded a putative plasma membrane di-heme protein in mouse duodenal mucosa. Dcytb shared between 45 and 50% similarity to the cytochrome b561 family of plasma membrane reductases, was highly expressed in the brush-border membrane of duodenal enterocytes, and induced ferric reductase activity when expressed in Xenopus oocytes and cultured cells. Duodenal expression levels of Dcytb messenger RNA and protein were regulated by changes in physiological modulators of iron absorption. Thus, Dcytb provides an important element in the iron absorption pathway.     

Rapid switching of plant gene expression induced by fungal elicitor

The pattern of messenger RNA synthesis in suspension-cultured bean cells (Phaseolus vulgaris L.) was analyzed by blot hybridization and in vitro translation of newly synthesized messenger RNA. The RNA was separated from preexisting RNA by organomercurial affinity chromatography after in vivo labeling with 4-thiouridine. The elicitor induced the synthesis of messenger RNA's encoding phenylalanine ammonia-lyase, chalcone synthase, and chalcone isomerase, three enzymes of phenylpropanoid metabolism involved in the synthesis of isoflavonoidderived phytoalexins. This is part of a rapid and extensive change in the pattern of messenger RNA synthesis directing production of a set of proteins associated with expression of disease resistance.     

Involvement of RNA in the synthesis of proteins

We can now have considerable confidence that the broad features of protein synthesis are understood. The involvement of RNA is very much more complicated than was imagined in 1953. There is not one functional RNA. Instead, protein synthesis demands the ordered interaction of three classes of RNA-ribosomal, soluble, and messenger. Many important questions, however, remain unanswered. For instance, there is no theoretical framework for the ribosomal subunits, nor for that matter, do we understand the functional significance of ribosomal RNA. Most satisfying is the realization that all the steps in protein replication will be shown to involve well-understood chemical forces. As yet we do not know all the details. For example, are the DNA base pairs involved in messenger RNA selection of the corresponding amino-acyl-sRNA? With luck, this will soon be known. We can thus have every expectation that future progress in understanding selective protein synthesis (and its consequences for embryology) will have a similarly well-defined and, when understood, easy-to-comprehend chemical basis (62).     

Gene transfer and expression of human phenylalanine hydroxylase

Phenylketonuria (PKU) is caused by a genetic deficiency of the enzyme phenylalanine hydroxylase (PAH). A full-length complementary DNA clone of human PAH was inserted into a eukaryotic expression vector and transferred into mouse NIH3T3 cells which do not normally express PAH. The transformed mouse cells expressed PAH messenger RNA, immunoreactive protein, and enzymatic activity that are characteristic of the normal human liver products, demonstrating that a single gene contains all of the necessary genetic information to code for functional PAH. These results support the use of the human PAH probe in prenatal diagnosis and detection of carriers, to provide new opportunities for the biochemical characterization of normal and mutant enzymes, and in the investigation of alternative genetic therapies for PKU.     

Ectopic pro-opiolipomelanocortin: sequence of cDNA coding for beta-melanocyte-stimulating hormone and beta-endorphin

A recombinant bacterial plasmid, pMS1, was constructed that contains 318 nucleotides complementary to a portion of pro-opiolipomelanocortin (proOLMC) messenger RNA from an ectopic adrenocorticotropin-producing tumor. The cloned complementary DNA insert, which contains the sequence that codes for all of the beta-melanocyte-stimulating hormone and beta-endorphin portions of proOLMC, as well as the 3' nontranslated section, is identical to the genomic sequence. Hybridization of tumor proOLMC complementary DNA to RNA subjected to electrophoresis and transferred to a nitrocellulose filter revealed two proOLMC messenger RNA species in the tumor polyadenylated RNA, but only one in pituitary polyadenylated RNA. At least one of the tumor proOLMC messenger RNA's is similar, if not identical, to human pituitary proOLMC messenger RNA.     

A single troponin T gene regulated by different programs in cardiac and skeletal muscle development

A cloned complementary DNA derived from a messenger RNA transiently present at low abundance levels in early chick embryonic skeletal muscle hybridizes to a messenger RNA present at high abundance levels in cardiac muscle. Genomic DNA hybridization and nucleotide sequence identity of complementary DNA's from both heart and skeletal muscle demonstrate that the messenger RNA's from both sources are encoded by the same gene. The encoded polypeptide is a troponin T sequence which is probably a cardiac isoform. This single copy troponin T isogene is governed by different regulatory programs in heart and skeletal muscle differentiation.     

R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway

The RNA interference (RNAi) pathway is initiated by processing long double-stranded RNA into small interfering RNA (siRNA). The siRNA-generating enzyme was purified from Drosophila S2cells and consists of two stoichiometric subunits: Dicer-2(DCR-2) and a previously unknown protein that we named R2D2. R2D2 is homologous to the Caenorhabditis elegans RNAi protein RDE-4. Association with R2D2 does not affect the enzymatic activity of DCR-2. Rather, the DCR-2/R2D2 complex, but not DCR-2 alone, binds to siRNA and enhances sequence-specific messenger RNA degradation mediated by the RNA-initiated silencing complex (RISC). These results indicate that R2D2 bridges the initiation and effector steps of the Drosophila RNAi pathway by facilitating siRNA passage from Dicer to RISC.     

Identification of virus-encoded microRNAs

RNA silencing processes are guided by small RNAs that are derived from double-stranded RNA. To probe for function of RNA silencing during infection of human cells by a DNA virus, we recorded the small RNA profile of cells infected by Epstein-Barr virus (EBV). We show that EBV expresses several microRNA (miRNA) genes. Given that miRNAs function in RNA silencing pathways either by targeting messenger RNAs for degradation or by repressing translation, we identified viral regulators of host and/or viral gene expression.     

Molecular cloning of two types of GAP complementary DNA from human placenta

The ras p21 GTPase-activating protein (GAP) was purified from human placental tissue. Internal amino acid sequence was obtained from this 120,000-dalton protein and, by means of this sequence, two types of complementary DNA clones were isolated and characterized. One type encoded GAP with a predicted molecular mass of 116,000 daltons and 96% identity with bovine GAP. The messenger RNA of this GAP was detected in human lung, brain, liver, leukocytes, and placenta. The second type appeared to be generated by a differential splicing mechanism and encoded a novel form of GAP with a predicted molecular mass of 100,400 daltons. This protein lacks the hydrophobic amino terminus characteristic of the larger species, but retains GAP activity. The messenger RNA of this type was abundantly expressed in placenta and in several human cell lines, but not in adult tissues.     

Expression of functional cell-cell channels from cloned rat liver gap junction complementary DNA

An oocyte expression system was used to test the relation between a complementary DNA (cDNA) clone encoding the liver gap junction protein and cell-cell channels. Total liver polyadenylated messenger RNA injected into oocytes induced cell-cell channels between paired oocytes. This induction was blocked by simultaneous injection of antisense RNA transcribed from the gap junction cDNA. Messenger RNA selected by hybridization to the cDNA clone and translated in oocyte pairs yielded a higher junctional conductance than unselected liver messenger RNA. Cell-cell channels between oocytes were also formed when the cloned cDNA was expressed under the control of a heat-shock promoter. A concentration-dependent induction of channels was observed in response to injection with in vitro transcribed gap junction messenger RNA. Thus, the liver gap junction cDNA encodes a protein that is essential for the formation of functional cell-cell channels.     

Expression of a calcium-mobilizing parathyroid hormone-like peptide in lactating mammary tissue

A survey of rat tissues by RNA analysis, aimed at uncovering the physiological function of the parathyroid hormone-like peptide (PTH-LP) associated with hypercalcemia of malignancy, revealed the presence of a 1.5-kilobase messenger RNA encoding this peptide in lactating mammary glands. PTH-LP messenger RNA is expressed in mammary tissue only during lactation; it appears and disappears rapidly (2 to 4 hours) as a function of the sucking stimulus. The identity of this messenger RNA was confirmed by cloning the rat PTH-LP complementary DNA, which predicts a peptide with strong similarity to the human homolog. Moreover, extracts from lactating mammary tissue stimulated parathyroid hormone-dependent adenylate cyclase. These findings suggest that PTH-LP plays a physiological role in lactation, possibly as a hormone for the mobilization or transfer (or both) of calcium to the milk.     

Molecular cloning of complementary DNA for the alpha subunit of the G protein that stimulates adenylate cyclase

A complementary DNA clone encoding the alpha subunit of the adenylate cyclase stimulatory G protein (Gs) was isolated and identified. A bovine brain complementary DNA library was screened with an oligonucleotide probe derived from amino acid sequence common to known G proteins. The only clone that was obtained with this probe has a complementary DNA insert of approximately 1670 base pairs. An antibody to a peptide synthesized according to deduced amino acid sequence reacts specifically with the alpha subunit of Gs. In addition, RNA that hybridizes with probes made from the clone is detected in wild-type S49 cells; however, cyc- S49 cells, which are deficient in Gs alpha activity, are devoid of this messenger RNA.     

Isolation of an olfactory cDNA: similarity to retinol-binding protein suggests a role in olfaction

Molecular cloning techniques were used to isolate and characterize a protein possibly involved in the signal transducing system in olfactory tissue of the frog Rana pipiens. A complementary DNA library was constructed with messenger RNA obtained from frog olfactory neuroepithelium. A 700-base pair complementary DNA clone encoding a protein with a molecular weight of 20,300 was identified by differential hybridization analysis with polyadenylated RNA from olfactory epithelium and nonsensory respiratory epithelium. The messenger RNA corresponding to this clone was abundant in the cells of Bowman's glands in olfactory tissue but not in respiratory epithelium nor in several other tissues. The predicted sequence of this protein is homologous to members of a family of proteins that bind and transport small molecules in serum, suggesting that this protein may also bind and transport odorants in the mucus secreted by Bowman's glands.     

Genetic variation in the human insulin gene

Four recombinant lambda phages containing nucleotide sequences complementary to a cloned human preproinsulin DNA probe have been isolated from human DNA. Restriction analyses in conjunction with Southern hybridizations reveal two types of gene sequences. One isolate of each type was subjected to complete nucleotide sequence determination. The sequences contain the entire preproinsulin messenger RNA region, two intervening sequence. 260 nucleotides upstream from the messenger RNA capping site, and 35 nucleotides beyond the polyadenylate attachment site. Our results strongly suggest that these two gene types are allelic variants of a single insulin gene.     

Human immunodeficiency virus may encode a novel protein on the genomic DNA plus strand

The genome of the human immunodeficiency virus (HIV) is known to contain eight open reading frames (ORFs) on the minus strand of the double-stranded DNA replicative intermediate. Data presented here indicate that the DNA plus strand of HIV contains a previously unidentified ORF in a region complementary to the envelope gene sequence. This ORF could encode a protein of approximately 190 amino acid residues with a relative molecular mass of 20 kilodaltons if translation began from the first initiation codon. The predicted protein is highly hydrophobic and thus could be membrane associated. It is possible, therefore, that the HIV genome encodes a protein on antisense messenger RNA.