Size Classes Of RNA Research Articles

Human Tat-specific factor 1 binds the HIV-1 genome and selectively transports HIV-1 RNAs.

Human immunodeficiency virus type 1 (HIV-1) propagation requires many human cofactors. Multiple groups have demonstrated that Tat-specific factor 1 (Tat-SF1) is an HIV-1 dependency factor. Depletion of this protein lowers HIV-1 infectivity, however, it does not affect the overall levels of viral RNA. Rather, Tat-SF1 regulates the relative levels of each RNA size class. This would be consistent with roles in splicing, transport, and/or stability of viral RNAs. We hypothesized that if Tat-SF1 plays any of these roles, then we should detect binding of the protein to the RNA genome. Furthermore, knocking down Tat-SF1 should result in altered RNA stability and/or localization in human cells. Fragments of the HIV-1 genome were used as RNA probes in electrophoretic mobility shift assays and fluorescence correlation spectroscopy experiments. Our results show that Tat-SF1 can form a complex with TAR RNA in vitro, independent of Tat. This factor interacts with at least one additional location in the 5' end of the HIV-1 genome.Tat seems to enhance the formation of this complex. To analyze HIV-1 RNA localization, HeLa cells with Tat-SF1 knocked down were also transfected with a proviral clone. RNA from nuclear and cytoplasmic fractions was purified, followed by RT-qPCR analysis. Tat-SF1 likely binds the HIV-1 RNA genome at TAR and potentially other locations andselectively transports HIV-1 RNAs, facilitating the unspliced RNA's nuclear export while retaining singly spliced RNAs in the nucleus. This is a novel role for this HIV-1 dependency factor.

Global Epigenetic and Transcriptional Trends among Two Rice Subspecies and Their Reciprocal Hybrids

The behavior of transcriptomes and epigenomes in hybrids of heterotic parents is of fundamental interest. Here, we report highly integrated maps of the epigenome, mRNA, and small RNA transcriptomes of two rice (Oryza sativa) subspecies and their reciprocal hybrids. We found that gene activity was correlated with DNA methylation and both active and repressive histone modifications in transcribed regions. Differential epigenetic modifications correlated with changes in transcript levels among hybrids and parental lines. Distinct patterns in gene expression and epigenetic modifications in reciprocal hybrids were observed. Through analyses of single nucleotide polymorphisms from our sequence data, we observed a high correlation of allelic bias of epigenetic modifications or gene expression in reciprocal hybrids with their differences in the parental lines. The abundance of distinct small RNA size classes differed between the parents, and more small RNAs were downregulated than upregulated in the reciprocal hybrids. Together, our data reveal a comprehensive overview of transcriptional and epigenetic trends in heterotic rice crosses and provide a useful resource for the rice community.

Developmentally-specific transcripts from the ccmFN-rps1 locus in wheat mitochondria

We have examined precursor and processed transcripts arising from the wheat mitochondrial ccmFN-rps1 region, which encodes a cytochrome c biogenesis component and S1 ribosomal protein, for the embryo-to-seedling stages of development. Northern analysis revealed 3.2-kb ccmFN-rps1 precursors, 2.6-kb bicistronic mRNA and 0.7-kb monocistronic rps1 transcripts, although their relative abundances were seen to shift during development. The 3.2-kb transcript levels peak during the 12-h to 2-day period, whereas 2.6-kb transcripts continue to increase during seedling growth, consistent with the newly-synthesized RNAs being more efficiently processed in later developmental stages. The 3.2-kb ccmFN-rps1 precursors consist of primary transcripts and 5'-processed RNAs based on pyrophosphatase-treated circular-RT-PCR analysis, whereas the 5' termini of 2.6-kb transcripts appear to be generated by endonucleolytic cleavage. The 0.7-kb rps1 transcripts are abundant during early germination but not in the seedlings; their 5' ends are heterogeneous and most of them lack the expected initiation codon. Notably all three size classes of RNAs share similar 3' termini. The 2.6-kb ccmFN-rps1 mRNAs exhibited full C-to-U editing at the sites examined, whereas the other two categories were slightly under-edited. A subset of all three-sized transcripts possessed short stretches of non-encoded adenosines, thus adding another layer of complexity to RNA level events in plant mitochondria.

Molecular genetics of the Drosophila melanogaster ovo locus, a gene required for sex determination of germline cells.

The Drosophila melanogaster ovo gene is required for survival and differentiation of female germline cells, apparently playing a role in germline sex determination. We recovered 60 kb of genomic DNA from its genetic location at 4E1,2 on the X chromosome. A transcription unit coding for an apparently female-specific germline-dependent 5-kb poly(A)+ RNA size class is located substantially in a 7-kb region, within which three DNA-detectable lesions for mutations that inactivate the ovo function are located at two sites approximately 4 kb apart. The breakpoint of a deficiency that removes the neighboring lethal complementation group shavenbaby (svb) but leaves the ovo function intact maps approximately 5 kb to the molecular left of the leftmost ovo mutant site. A class of mutations that inactivates both the svb function and the ovo function affects genomic DNA between the two ovo sites. Sequences required for the two genetic functions are partly overlapping. In spite of this overlap, P element-mediated gene transfer of a 10-kb genomic DNA segment containing the 5-kb poly(A)+ RNA transcription unit rescues the female sterility phenotypes of ovo mutations, but not the svb lethality.

Thermal stability of turnip yellow mosaic virus RNA: effect of pH and multivalent cations on RNA deaggregation and degradation

Light scattering studies of RNA isolated from turnip yellow mosaic virus (TYMV) revealed a molar mass of 1.9.10(6) g mol-1, which is close to the value of 2.0.10(6) g mol-1 published for intact genomic TYMV RNA (2M RNA). However, gel electrophoresis under denaturing conditions demonstrated that only 30-40% of this native RNA was 2M RNA. Sucrose gradient centrifugation revealed the occurrence of a series of smaller RNA size classes, the mass ratios of which were greatly influenced by the pH of the solution and the presence of EDTA. These results suggest that native TYMV RNA preparations originally contain a mixture of intact RNA particles and of aggregates of RNA fragments with the same molar mass of about 2.10(6) g mol-1, and that the size classes are intermediates in the deaggregation process of the degraded genomic TYMV RNA. The native RNA displayed pH-dependent deaggregation and degradation. The degradation process of 2M RNA followed (pseudo) first-order kinetics. Lower degradation rates were observed for RNA depleted of divalent cations and polyamines. For depleted 2M RNA an enthalpy of activation of about 100 kJ mol-1 and an almost zero entropy of activation was calculated. Similar values were also found for depleted E. coli ribosomal RNAs and depleted MS2 RNA, demonstrating that all RNAs are equally vulnerable to degradation. In the presence of multivalent cations the activation enthalpy for 2M TYMV RNA degradation increased to 150 kJ mol-1 and the entropy of activation to 150 J K-1 mol-1, indicative for a different degradation mechanism.

Characterization of the human N-CAM promoter

In contrast with the complex series of splicing choices that generate the various membrane-associated isoforms of the neural cell-adhesion molecule alternative splicing of 5' exons does not contribute to additional molecular diversity. A single regulatory unit in genomic DNA, mapping to a 5 kb restriction-endonuclease-HindIII fragment, controls the expression of all major RNA size classes. DNA sequence analysis of a 2 kb fragment spanning the two major identified transcriptional initiation sites (194 and 188 bp from the ATG codon) and translation start codon indicates that the regulatory unit does not possess classical TATA or CCAAT motifs. The region of the putative promoter exhibits a GC-rich content and a high frequency of the dinucleotide CpG, both characteristics of a HTF(HpaII tiny fragments)-island. Introduction of deletion-mutant chimaeric-gene constructs into human and rodent N-CAM-expressing cell lines defines an active promoter region of 467 bp (-144 to -611 bp from the ATG codon). This region of genomic DNA contains consensus sites for the interaction of known transcriptional factors.

An insertion of insect cell DNA in the 81-map-unit segment of Autographa californica nuclear polyhedrosis virus DNA

In this report, a transposonlike insertion of Spodoptera frugiperda insect cell DNA was analyzed in single-plaque isolate E of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). The 634-base-pair insertion is characterized by an 18-base-pair terminal inverted repeat and carries an EcoRI site. This additional EcoRI site in the 81-map-unit segment of the DNA of plaque isolate E of AcNPV explains the difference between the EcoRI restriction map of the DNA from this isolate and those of the virus stocks used in other laboratories. Except for this insertion, the nucleotide sequence at the site of insertion in the DNA of plaque isolate E is identical to that of AcNPV E2 (G. E. Smith and M. D. Summers, Virology 89:517-527, 1978). The cellular DNA insertion in the AcNPV genome is represented many times in the S. frugiperda cell genome but has no detectable homology with DNAs from species other than lepidopteran insects. In S. frugiperda cells, the transposonlike insertion sequences are transcribed into cytoplasmic RNA. The transcription of these sequences is initiated within the cellular insertion element. As reported previously (C. Oellig, B. Happ, T. Müller, and W. Doerfler, J. Virol. 61:3048-3057, 1987), in S. frugiperda cells infected with plaque isolate E of AcNPV, at least nine different size classes of AcNPV-specific RNAs are synthesized; in AcNPV E2-infected cells, similar size classes have been detected. The cellular insertion of plaque isolate E provides the initiation site for the synthesis of an additional RNA size class which is transcribed off viral DNA.

Characterization of INT-2: A member of the fibroblast growth factor family

int-2 was discovered as a proto-oncogene transcriptionally activated by MMTV proviral insertion during mammary tumorigenesis in the mouse. Sequence analysis showed int-2 to be a member of the fibroblast growth factor family of genes. In normal breast and most other adult mouse tissues, int-2 expression was not detected except for low levels in brain and testis. However, using in situ hybridization, expression was found at a number of sites during embryonic development, from day 7 until birth. An analysis of the int-2 transcripts found in embryonal carcinoma cells revealed six major classes of RNA initiating at three promoters and terminating at either of two polyadenylation sites. Despite the transcriptional complexities, all size classes of RNA encompass the same open reading frame. Using an SV40 early promoter to drive transcription of an int-2 cDNA in COS-1 cells, several proteins were observed. These were shown to be generated by initiation from either of two codons: One, a CUG, leads to a product which localizes extensively to the cell nucleus and partially to the secretory pathway. In contrast, initiation at a downstream AUG codon results in quantitative translocation across the endoplasmic reticulum and the accumulation of products ranging in size from 27.5 x 10(3) Mr to 31.5 x 10(3) Mr in organelles of the secretory pathway. These proteins represented glycosylated and non-glycosylated forms of the same primary product with or without the signal peptide removed. These findings suggest the potential for a dual role of int-2; an autocrine function acting at the cell nucleus, and a possible paracrine action through a secreted product.

DNA sequence and regulation of the gene (cbpA) encoding the 42-kilodalton cytoplasmic membrane carotenoprotein of the cyanobacterium Synechococcus sp. strain PCC 7942

The gene (cbpA) coding for a carotenoid-binding protein of the cyanobacterium Synechococcus sp. strain PCC 7942 (Anacystis nidulans R2) has been cloned and sequenced. A polyclonal antibody against the protein was used to identify immunoreactive clones from a lambda gt11 expression library of Synechococcus strain PCC 7942. The initial positive clone (lambda gtAN42) contained a 0.9-kilobase (kb) chromosomal fragment, which was used to detect a larger chromosomal fragment from a lambda EMBL3 library. The lambda EMBL3 recombinant, lambda EM109, contained an 18-kb portion of the Synechococcus strain PCC 7942 chromosome. The open reading frame of cbpA encoded 450 amino acids which give rise to a protein of 49,113 daltons. The hydrophobicity plot indicates that the protein may have a 49-residue signal sequence which is cleaved to yield a mature protein of 43,709 daltons. The protein has been localized in the cytoplasmic membrane by biochemical procedures as well as by electron microscopic immunocytochemistry. Northern (RNA) blot analysis indicates that transcription of cbpA is tightly regulated by DNA topology, light intensity, and iron concentration. Transcription is greatly induced by growth under high light intensities and repressed during growth under iron-deficient conditions. The DNA gyrase inhibitor novobiocin specifically inhibited the light-induced transcription. In Northern blots, the gene-specific probe hybridized to two size classes of RNA, with lengths of 2.0 and 6.2 kb. Since cbpA appears to be a component of the 6.2-kb transcript, it is likely part of a larger operon.

Maintenance by erythropoietin of viability and maturation of murine erythroid precursor cells

Erythroblasts isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus (FVA cells)-are erythropoietin (EP)-sensitive cells at the late colony forming unit-erythroid (CFU-E) and cluster forming unit stages of differentiation (Koury et al., J. Cell. Physiol. 121:526-532, 1984). We investigate here the EP requirements of FVA cells in vitro for viability, proliferation, and maturation. By delaying the addition of EP to FVA cell cultures or by withdrawing EP at early times of culture, the subsequent viability, cell numbers, and maturation were diminished. The longer the delay in EP addition or the earlier the EP withdrawal, the more diminished these parameters were when compared to cultures which contained EP throughout the 48 h of differentiation. FVA cells had a period of EP requirement in vitro that lasted for only 24 h or less after the initiation of culture. During these crucial first 24 h, EP induced an increase in the synthesis of all size classes of RNA. Protein synthesis was maintained at a stable level in cells cultured with EP, but it declined in cells cultured without it. In contrast, the synthesis rate of DNA and the content of DNA per cell were not affected by the presence of EP in the culture. However, FVA cells cultured without EP had progressive accumulation of small sized DNA due to breakage of higher molecular weight DNA. The rate of DNA breakdown was sufficient to prevent DNA accumulation and thus it probably plays a role in the abortion of cell proliferation. No such breakage was found in cells cultured with EP. Our results indicate that EP exerts an effect on FVA cells in culture which is reflected in their viability, cell number, and maturation. This effect is not mediated by a stimulation of the rate of DNA synthesis, but is accompanied by stimulation of overall RNA synthesis and maintenance of protein synthesis.

Effects of corticotropin-releasing hormone and dexamethasone on proopiomelanocortin messenger RNA level in human corticotroph adenoma cells in vitro.

The effects of corticotropin-releasing hormone (CRH) and dexamethasone on proopiomelanocortin (POMC) mRNA levels in cultured pituitary adenoma cells were studied in 10 patients with Cushing's disease. As a control, POMC mRNA levels in cells from nonadenomatous tissues were examined in four patients. Human POMC mRNA in the cells was analyzed by Northern blot hybridization. Human POMC DNA probe hybridized with only a single size class of RNA (approximately 1,200 nucleotides) from the adenoma and nonadenoma cells of each patient. The size of POMC mRNA did not change through the culture or after incubation with CRH or dexamethasone. CRH increased POMC mRNA levels in these cells in a dose- and time-dependent manner. The minimum concentration of CRH required to elevate POMC mRNA levels in these cells exposed for 15 h was 0.1 nM. The minimum duration of 1 nM CRH treatment required to increase these levels was 3 h under our conditions. Inhibitory effects of 1 and 10 micrograms/dl dexamethasone on ACTH release and POMC mRNA levels in nonadenoma cells were greater than those in adenoma cells. These results suggest the following: (a) that the mRNA in cultured pituitary adenoma cells is qualitatively the same as that in vivo; (b) that responses of mRNA levels to CRH are time- and dose-dependent; and (c) that adenoma cells resist the inhibitory effect of dexamethasone on POMC mRNA levels and ACTH release.

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.

Overlapping sets of viral RNAs reflect the array of polypeptides in the EcoRI J and N fragments (map positions 81.2 to 85.0) of the Autographa californica nuclear polyhedrosis virus genome

In several parts of the Autographa californica nuclear polyhedrosis virus (AcNPV) genome, nested sets of overlapping RNAs with common 3' or 5' termini have been recognized. In the present report, the pattern of viral transcription and the arrangement of viral gene products in the region of 81.2 to 85.0 map units were investigated. In this segment of the AcNPV genome, at least nine size classes of viral RNA were identified which ranged in size from 1.3 kilobases (kb) to 4.6 kb and exhibited common 3' termini. The detailed restriction map and the nucleotide sequence of this part of the AcNPV genome were determined. Computer analyses revealed several open reading frames (ORFs) on the rightward-transcribed strand with potential TATA and CAAT signals preceding many of the potential ORFs and the 5' termini of some of the mapped RNAs. The leftward-transcribed strand was devoid of major ORFs. The presumptive polypeptides encoded by the larger ORFs ranged in size from 11.3 to 55.6 kilodaltons (kDa). The amino acid sequence of the presumptive polypeptide encoded by ORF3, a 33.6-kDa molecule, exhibited an unusual, clustered 16-fold repeat of the dipeptide arginine-serine in a protein that showed an overall preponderance of basic amino acids. The results of in vitro translation experiments with hybrid-selected RNAs homologous to internal subfragments of the 81.2- to 85.0-map-unit region yielded polypeptides of approximately 28, 34 to 36, and 48 to 50 kDa, which were close in size to the lengths of the major ORFs derived from the nucleotide sequence. The localizations of individual size classes of RNAs in the 81.2- to 85.0-map-unit region of the viral genome were determined precisely at the 3' and 5' termini by S1 protection analyses. Within a sequence of eight nucleotides, all RNAs had the same 3' terminus, which lay close to multiple polyadenylation signals. The initiation sites of the nine different RNA size classes were precisely mapped. As the cap sites of the smaller RNAs (less than 1.8 kb) were determined by S1 protection analyses, a multitude of RNA initiation sites became apparent. It was also shown that the different RNA size classes in the 81.2- to 85.0-map-unit region were detectable as early as 2 h and at least until 36 to 48 h after infection. In unselected cytoplasmic RNA, the size classes of viral RNAs specific for the EcoRI J fragment were detectable early as well as late after infection, although at early times the larger RNAs were detectable in smaller amounts.(ABSTRACT TRUNCATED AT 400 WORDS)

MRNAs coding for proteins of the cGMP cascade in the degenerative retina of the rd mouse

A lesion in cGMP metabolism has been hypothesized to cause retinal degeneration in rd mice. Available cloned cDNAs coding for proteins involved in the cGMP cascade have been used to compare the corresponding retinal RNAs in the degenerative (rd/rd) mouse at 8-11 days with those in the 8-11-day-old morphologically normal (rd/+) and adult normal (+/+) mice. Northern analysis of these RNAs hybridized to the specific 32P-labeled cDNA probes for G-protein, 48,000 MW protein and opsin, indicates in each case, that the corresponding transcripts are made in the rd/rd mouse retina and that there are no overt differences in their size compared to the transcripts hybridized in the rd/+ or +/+ mouse retinas. Although a defect in the phosphorylation of opsin has been described in rd mice, no difference was found in the transcripts hybridized by a cDNA probe corresponding to the region of the opsin molecule on which phosphorylation occurs. We do find, however, that labeled bovine-derived opsin cDNA recognizes five different RNA size classes in mouse and bovine retinas. Control experiments were performed to confirm that the RNA hybridized by opsin cDNA was not due to non-specific hybridization to unrelated RNAs.

The lethal yellow allele-associated provirus results in the production of chimeric viral-host RNAs.

The lethal yellow (Ay) mutation is the most dominant allele at the agouti locus of the mouse. Mice heterozygous for this allele have a yellow coat color, are genetically obese with an increased susceptibility to cancer, and have other metabolic derangements. Mice homozygous for Ay die early in embryogenesis, possibly because of a trophectoderm defect. The Ay mutation is distinguished from the many alleles described at the agouti locus in being associated with an endogenous provirus, designated endogenous ecotropic murine leukemia viral locus 15 (Emv-15). To obtain DNA sequences from regions close to or within the agouti locus, we have isolated the Emv-15 provirus and have found that the DNA sequences adjacent to the provirus are part of a mouse gene that is expressed in the same transcriptional orientation as the proviral genes. These mouse DNA sequences recognize two distinct size classes of RNA in various adult wild-type tissues including skin. In Ay heterozygotes both types of transcript hybridize to the proviral long terminal repeat, and, in heterozygous spleens, the shorter transcript is present at an enhanced steady-state level. These results suggest that the Ay-associated provirus (Emv-15) is distinguished from the endogenous C-type proviruses described to date in having altered the expression of a flanking host gene by promoter insertion, resulting in the production of chimeric viral-host fusion transcripts.

Rat brain 5-HT1C receptors are encoded by a 5-6 kbase mRNA size class and are functionally expressed in injected Xenopus oocytes

Injection of rat brain RNA into Xenopus laevis oocytes induces synthesis of receptors that show an electrophysiological response to bath application of serotonin. While there are at least 4 pharmacologically distinct subtypes of 5-HT binding sites in the rat brain, we find that the pharmacological characteristics of the predominant electrophysiologically active receptor synthesized in Xenopus oocytes are most consistent with those of the 5-HT1C subtype. Additional electrophysiologically active 5-HT receptor types could not be detected. Injection of mRNA isolated from a number of rat brain regions shows that the choroid plexus is particularly enriched for 5-HT1C mRNA. Oocytes injected with RNA isolated from this region respond 16 or 8 times more strongly to serotonin than do oocytes injected with RNA isolated from cortex or substantia nigra, respectively. In addition, by fractionation of rat brain mRNA through agarose gels, we have identified a single RNA size class of about 5-6 kbase that encodes this serotonin receptor.

The human U1-70K snRNP protein: cDNA cloning, chromosomal localization, expression, alternative splicing and RNA-binding

We have isolated and sequenced cDNA clones encoding the human U1-70K snRNP protein, and have mapped this locus (U1AP1) to human chromosome 19. The gene produces two size classes of RNA, a major 1.7-kb RNA and a minor 3.9-kb RNA. The 1.7-kb species appears to be the functional mRNA; the role of the 3.9-kb RNA, which extends further in the 5' direction, is unclear. The actual size of the hU1-70K protein is probably 52 kd, rather than 70 kd. The protein contains three regions similar to known nucleic acid-binding proteins, and it binds RNA in an in vitro assay. Comparison of the cDNA sequences indicates that there are multiple subclasses of mRNA that arise by alternative pre-mRNA splicing of at least four alternative exon segments. This suggests that multiple forms of the hU1-70K protein may exist, possibly with different functions in vivo.

The ubiquitin genes in D. melanogaster: Transcription and polymorphism

We report here the identification, transcription and polymorphism of ubiquitin genes in Drosophila melanogaster. Ubiquitin is the only product of a tandemly arranged ‘repeat’ mapping at two closely linked sites within the early ecdysone and heat-shock puff region 63F. Complementary RNA to cloned 63F sequences can be translated in the in vitro reticulocyte system to give a protein product with a molecular weight of 8500 that comigrates with purified insect ubiquitin. The nucleotide sequence of the 63F repeat of Drosophila revealed that the protein product has amino-acid sequences identical to the ubiquitin proteins described for other organisms. There are probably two polyubiquitin genes per haploid genome in Drosophila, as detected by Southern blot analysis, one of which is highly polymorphic. The transcript pattern is quite complex, with four (sometimes five) RNA size classes all very polymorphic except for one of 0.9 kb present all through development and highly conserved in different stocks and strains. We suggest that unequal crossing-over is the mechanism generating the observed variations in the ubiquitin genes of D. melanogaster.

Some biological effects of electromagnetic fields

It has been known for some time that specific asymmetric electromagnetic fields can induce union in non-healing bones. Despite the clinical effectiveness of low frequency electromagnetic (e.m.) signals, virtually nothing is known concerning basic mechanisms in cells involved in bone healing, or other cells in soft tissue, which can also be affected by e.m. signals. It has thus become increasingly important to understand the molecular basis of e.m. field stimulation in order to evaluate the clinical effectiveness and the results of inadvertent environmental exposure. We have developed a test system to examine a fundamental cellular function, that of transcription, in Sciara polytene chromosomes. Since RNA synthesis is a basic cellular event, alterations arising from exogenous stimulation of cells by e.m. fields should be reflected in transcriptional patterns. Our results show this to be the case. e.m. signals in the 72 Hz frequency range, both quasi-rectangular and sinusoidal, induce new RNA transcription in the 6–10S size class and augment a RNA size class in the 20–25S range. e.m. signals in the 1.5 to 15 Hz frequency range result in either a similar response as that seen in 72 Hz signals, or one which is indistinguishable from non-stimulated control cells. Since translational effects would be expected to be reflected as a result of alterations in transcription, we examined protein biosynthesis following e.m. field stimulation. Changes in the form of augmentation, deletion, and appearance of new polypeptides are observed. Finally, we found that e.m. signals, even at some distance from the non-stimulated control samples, emit an undefined signal that is detected by the non-stimulated control cells. For this reason, we have shielded non-stimulated cells. Our ultimate goal is to correlate a specific parameter of the e.m. signal waveform with cellular induction of particular classes of RNA and/or molecular weight ranges of protein. To date, the data suggest that the 72 Hz range is an important frequency component and that waveshape may not be as crucial to clinical usefulness as previously supposed.

Multiple mRNAs for human alcohol dehydrogenase (ADH): developmental and tissue specific differences.

Human class I alcohol dehydrogenase (ADH) genes show developmental and tissue specific differences in expression at the polypeptide level. In these studies ADH expression was investigated at the RNA level. Northern blot analysis of total and poly (A) RNA from adult liver using pADH12 probe demonstrated multiple RNA size classes of 2.6, 2.2, 1.9 and 1.6kb. In contrast, fetal liver, and fetal intestine contained only 2.6 and 1.6kb mRNA while fetal lung showed only 2.6kb mRNA. All of these tissues showed a relative reduction in the amount of ADH mRNA present when compared to adult liver. Immunoprecipitation of in vitro translation products of adult liver RNA by polyclonal ADH antibody revealed a single polypeptide of 40,000 daltons. This result points out the homogeneity of size of class I ADH polypeptides despite mRNA size diversity. Variation in length of the 3' untranslated region probably contributes to the multiple size classes of ADH mRNA observed.