Localization of amyloid beta protein messenger RNA in brains from patients with Alzheimer's disease

The distribution of cells containing messenger RNA that encodes amyloid beta protein was determined in hippocampi and in various cortical regions from cynomolgus monkeys, normal humans, and patients with Alzheimer's disease by in situ hybridization. Both 35S-labeled RNA antisense and sense probes to amyloid beta protein messenger RNA were used to ensure specific hybridization. Messenger RNA for amyloid beta protein was expressed in a subset of neurons in the prefrontal cortex from monkeys, normal humans, and patients with Alzheimer's disease. This messenger RNA was also present in the neurons of all the hippocampal fields from monkeys, normal humans and, although to a lesser extent in cornu ammonis 1, patients with Alzheimer's disease. The distribution of amyloid beta protein messenger RNA was similar to that of the neurofibrillary tangles of Alzheimer's disease in some regions, but the messenger RNA was also expressed in other neurons that are not usually involved in the pathology of Alzheimer's disease.     

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.     

Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum

The role of myosin in the contraction of striated muscle cells is well known, but its importance in nonmuscle cells is not yet clear. The function of myosin in Dictyostelium discoideum has been investigated by isolating cells which specifically lack myosin heavy chain (MHC A) protein. Cells were transformed with a vector encoding RNA complementary to mhcA messenger RNA (antisense RNA). Stable transformants have a dramatic reduction in the amount of MHC A protein, grow slowly, and generate giant multinucleated progeny, indicating an impairment in cytokinesis. Surprisingly, the cells adhere to surfaces, extend pseudopods and are capable of ameboid locomotion. The developmental sequence that is initiated by starving cells is severely impaired by the lack of myosin. The cells are unable to form multicellular aggregates normally and do not undergo subsequent morphogenesis. By changing the food source from liquid medium to bacteria, expression of the endogenous mhcA messenger RNA can be increased relative to expression of antisense RNA. When grown in this way, the transformed cells accumulate MHC A protein, remain mononucleate, and proceed through development normally.     

Probing gene expression in live cells, one protein molecule at a time

We directly observed real-time production of single protein molecules in individual Escherichia coli cells. A fusion protein of a fast-maturing yellow fluorescent protein (YFP) and a membrane-targeting peptide was expressed under a repressed condition. The membrane-localized YFP can be detected with single-molecule sensitivity. We found that the protein molecules are produced in bursts, with each burst originating from a stochastically transcribed single messenger RNA molecule, and that protein copy numbers in the bursts follow a geometric distribution. The quantitative study of low-level gene expression demonstrates the potential of single-molecule experiments in elucidating the workings of fundamental biological processes in living cells.     

Expression and characterization of the trans-activator of HTLV-III/LAV virus

The human T-lymphotropic retrovirus HTLV-III/LAV encodes a trans-activator that increases viral gene expression. We expressed this trans-activator in animal cells and studied its structural and functional characteristics. The putative trans-activator protein was immunoprecipitated from overproducing stable cell lines and shown to migrate as a 14-kilodalton polypeptide on sodium dodecyl sulfate-polyacrylamide gels. S1 nuclease mapping experiments showed that the trans-activator increases the levels of steady-state messenger RNA transcribed from the viral long terminal repeat promoter. Sequences within the R region of the HTLV-III/LAV long terminal repeat are essential for trans-activation. Quantitations of messenger RNA and protein showed that the protein increase was greater than the messenger RNA increase in CV1 and HeLa cells, indicating that more than one mechanism was responsible for the trans-activation and that cell type-specific factors may determine the final level of trans-activation.     

Identification of a peptide specific for Aplysia sensory neurons by PCR-based differential screening.

In order to identify genes specific for the sensory neurons of Aplysia, a miniaturized differential screening method based on the polymerase chain reaction and applicable to small amounts of tissue was used. One messenger RNA was isolated that is expressed in every mechanoreceptor sensory cluster of the Aplysia central nervous system. This messenger RNA encodes a peptide that seems to function as an inhibitory cotransmitter. The peptide selectively inhibits certain postsynaptic cells but not others and thereby allows the sensory neurons to achieve target-specific synaptic actions.     

Pertussis toxin gene: nucleotide sequence and genetic organization

The current pertussis vaccines, although efficacious, in some instances produce undesirable side effects. Molecular engineering of pertussis toxin, the major protective antigen, could provide a safer, new generation of vaccines against whooping cough. As a first critical step in the development of such a vaccine, the complete nucleotide sequence of the pertussis toxin gene was determined and the amino acid sequences of the individual subunits were deduced. All five subunits are coded by closely linked cistrons. A promoter-like structure was found in the 5'-flanking region, suggesting that the toxin is expressed through a polycistronic messenger RNA. The order of the cistrons is S1, S2, S4, S5, and S3. All subunits contain signal peptides of variable length. The calculated molecular weights of the mature subunits are 26,024 for S1, 21,924 for S2, 21,873 for S3, 12,058 for S4, and 11,013 for S5. Subunits S2 and S3 share 70% amino acid homology and 75% nucleotide homology. Subunit S1 contains two regions of eight amino acids homologous to analogous regions in the A subunit of both cholera and Escherichia coli heat labile toxins.     

Unusual pattern of accumulation of mRNA encoding EGF-related protein in sea urchin embryos

A sea urchin (Strongylocentrotus purpuratus) messenger RNA encoding a protein (SpEGF2) related to epidermal growth factor (EGF) was identified. The full-length complementary DNA sequence predicts a protein with an unusually simple structure, including four tandem EGF-like repeats and a hydrophobic leader, but lacking a potential transmembrane domain. Sequence similarities suggest that the peptides are homologous to two peptides from a different sea urchin species, which cause a classic developmental defect, exogastrulation, when added to the seawater outside of embryos. The SpEGF2 messenger RNA begins to accumulate at blastula stage, and in pluteus larvae it is distributed in discrete regions of ectoderm that are not congruent with known histological borders. One region corresponds to that expressing the homeodomain-containing protein, SpHbox1. The structure of the SpEGF2 protein and the pattern of accumulation of its messenger RNA suggest that it may have important functions as a secreted factor during development of sea urchin embryos.     

Antibodies to human c-myc oncogene product: evidence of an evolutionarily conserved protein induced during cell proliferation

Antisera to a synthetic c-myc peptide and to c-myc antigens synthesized from various portions of the human gene expressed in Escherichia coli were used in order to characterize the protein product of the human c-myc oncogene. Although the deduced molecular weight of the human c-myc protein is 49,000, these antisera precipitate a protein from human cells that migrates in sodium dodecyl sulfate-polyacrylamide gel as if its molecular weight were 65,000. In addition, the mouse c-myc protein, whether synthesized in cells or in a cell-free system directed by pure, synthetic messenger RNA, has analogous properties and is immunoprecipitated by the antiserum to the human c-myc protein. Similar proteins are immunoprecipitated from monkey, rat, hamster, and frog cells, suggesting evolutionary conservation of antigenic structure of the c-myc protein among vertebrates. In addition, and in a manner consistent with the behavior of its messenger RNA, the immunoprecipitable c-myc protein is sharply induced by the action of mitogens on resting human T cells.     

Viral persistence in neurons explained by lack of major histocompatibility class I expression

Viruses frequently persist in neurons, suggesting that these cells can evade immune surveillance. In a mouse model, 5 x 10(6) cytotoxic T lymphocytes (CTLs), specific for lymphocytic choriomeningitis virus (LCMV), did not lyse infected neurons or cause immunopathologic injury. In contrast, intracerebral injection of less than 10(3) CTL caused disease and death when viral antigens were expressed on leptomeningeal and choroid plexus cells of the nervous system. The neuronal cell line OBL21 expresses little or no major histocompatibility (MHC) class I surface glycoproteins and when infected with LCMV, resisted lysis by virus-specific CTLs. Expression of MHC heavy chain messenger RNA was limited, but beta 2-microglobulin messenger RNA and protein was made normally. OBL21 cells were made sensitive to CTL lysis by transfection with a fusion gene encoding another MHC class I molecule. Hence, neuronal cells probably evade immune surveillance by failing to express MHC class I molecules.     

Reversal of creatine kinase translational repression by 3' untranslated sequences

A subline of U937 cells (U937D) was obtained in which creatine kinase B (CK-B) messenger RNA was present and bound to ribosomes, but CK activity was undetectable. Transformation of U937D cells with retrovirus vectors that contain the 3' untranslated region (3' UTR) of CK-B messenger RNA exhibited CK activity with no change in abundance of CK-B mRNA. The 3' UTR formed a complex in vitro with a component of S100 extracts from wild-type cells. This binding activity was not detectable in S100 extracts from cells that expressed CK activity after transformation with the 3' UTR-containing vector. These results suggest that translation of CK-B is repressed by binding of a soluble factor or factors to the 3' UTR.     

Transcription of novel open reading frames of AIDS retrovirus during infection of lymphocytes

The retrovirus frequently isolated from patients with the acquired immune deficiency syndrome (AIDS) has two novel open reading frames previously designated "A" and "B." The "A" region was found to be specifically expressed as polyadenylated RNA's of 5.5 and 5.0 kilobases in infected cells. The "B" region was expressed as 1.8- to 2.0-kilobase RNA species. Additional full-length and spliced messenger RNA's of the env region were also identified.     

The 30-kilodalton gene product of tobacco mosaic virus potentiates virus movement

The proposed role of the 30-kilodalton(kD) protein of tobacco mosaic virus is to facilitate cell-to-cell spread of the virus-during infection. To directly define the function of the protein, a chimeric gene containing a cloned complementary DNA of the 30-kD protein gene was introduced into tobacco cells via a Ti plasmid-mediated transformation system of Agrobacterium tumefaciens. Transgenic plants regenerated from transformed tobacco cells expressed the 30-kD protein messenger RNA and accumulated 30-kD protein. Seedlings expressing the 30-kD protein gene complemented the Lsl mutant of TMV, a mutant that is temperature-sensitive in cell-to-cell movement. In addition, enhanced movement of the Lsl virus at the permissive temperature was detected in seedlings that express the 30-kD protein gene. These results conclusively demonstrate that the 30-kD protein of tobacco mosaic virus potentiates the movement of the virus from cell to cell.     

Hormonal induction of increased zinc uptake in mammalian cell cultures: requirement for RNA and protein synthesis

The zinc content of HeLa S(3) cells is markedly increased after growth in medium containing adrenal glucocorticoid hormones. Studies with inhibitors indicate that the synthesis of RNA and protein is required for enhanced zinc uptake. When protein synthesis is blocked in the presence of the steroid, an intermediate, presumably messenger RNA, which specifies enhanced zinc uptake accumulates and is expressed when the inhibition of protein synthesis is removed.