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Two pyrimidine alpha-LNA nucleoside monomers have been synthesised and incorporated into alpha-configured oligonucleotides. A fully modified mixed alpha-LNA sequence displays unprecedented parallel stranded hybridisation with complementary RNA and a remarkable selectivity for RNA over DNA. Modelling shows alpha-LNA:RNA to form an extended duplex with a very broad major groove.
A stereoregular all-(Sp)-boranophosphate oligodeoxyribonucleotide (BH3(-)-ODN) 15-mer was synthesized using an enzymatic approach. The BH3(-)-ODN formed a hybrid with the complementary RNA 15-mer and induced RNase H hydrolysis of the RNA strand at ODN concentrations as low as 10 nM at 37 degrees C, but with a lower efficiency than that of its natural phosphodiester analogue.
In situ hybridization is used to survey the tissue-specific and developmental expression of the cloned mouse gene Sparc, coding for a protein homologous to the bovine Ca++-binding protein, osteonectin. High levels of SPARC RNA are found in osteoblasts and odontoblasts. In addition, high grain counts are associated with a variety of other cell types in the embryo and newborn mouse, including parietal endoderm, deciduum, whisker follicles (connective tissue sheath), peripheral nerve trunk, skin (dermis), and stomach (submucosa). Spatially restricted but high levels of SPARC mRNA are also seen in the adult adrenal glands, testis, and ovary. This pattern of differential gene expression demands a reassessment of the function originally proposed for osteonectin, and predicts a much wider role for the protein in a variety of biological processes.
The repetitive DNA sequences of the newt, Triturus viridescens, have been studied by nucleic acid hybridization procedures. Complementary RNA was synthesized enzymatically from unfractionated newt DNA. This RNA hybridized strongly to the centromeric regions of both somatic and lampbrush chromosomes It also bound to other loci scattered along the lengths of the chromosomes The amplified ribosomal DNA in the multiple oocyte nucleoli was demonstrated by in situ hybridization
Locked nucleic acid (LNA) is a nucleic acid analog with very high affinity to complementary RNA and a promising compound in the field of antisense research. The intracellular localization and quantitative uptake of oligonucleotides containing LNA were found to be equivalent to those of phosphorothioate oligonucleotides (PS AONs). The antisense efficiency of LNA-containing oligonucleotides was systematically compared with standard PS AONs targeting expression of two endogenous proteins in the human breast cancer cell line MCF-7, namely, the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and the estrogen receptor alpha (ERalpha). For downregulation of both target proteins, the most efficient design was achieved with oligonucleotides containing LNA monomers in the extremities and a central gap of PS-linked DNA monomers, so called LNA gapmers. Such LNA gapmers caused more potent downregulation of the targeted proteins than PS AONs, whereas fully modified LNA AONs or LNA mixmers (LNA nucleotides interspersed) were inactive.
The c-myc proto-oncogene is a reliable marker of the "G0-early G1" transition, and its down-regulation is believed to be necessary to obtain cellular differentiation. In murine spermatogenesis, the level of c-myc transcripts does not correlate with the rate of cellular division. Proliferation of supposed staminal spermatogonia to reproduce themselves is induced with a local 5 Gy X-ray dose in 90-day-old C57Bl/6 mice. c-myc quantification by a newly developed competitive reverse transcriptase polymerase chain reaction (RT-PCR) was carried out to follow the expression course of this proto-oncogene. Damage and restoration of spermatogenesis were analyzed at days 3, 6, 9, 10, 13, 30, and 60 after injury by relative testes/body weight determination and histological examination. Proliferative status was determined by histone H3 Northern blot analysis. c-myc mRNA level was 10 times higher after 3 days in the irradiated animals compared to the controls. An increasing number of copies were noted up to 10 days, but promptly decreased to the base level found for irradiated mice from 13 to 60 days. Interestingly, the expression of histone H3 detected S phase only in testes at 60 days from damage.
The influenza virus RNA polymerase is a heterotrimer comprising the PB1, PB2 and PA subunits. PB1 is the core of the complex and accounts for the polymerase activity. We have studied the interaction of PB1 with model cRNA template by in vitro binding and Northwestern analyses. The binding to model cRNA was specific and showed an apparent Kd of approximately 7x10(-8) M. In contrast to the interaction with vRNA, PB1 was able to bind equally the 5' and 3' arm of the cRNA panhandle. The N-terminal 139 amino acids of PB1 and sequences between positions 267 and 493 proved positive for binding to cRNA, whereas the interaction with vRNA template previously was mapped to the N- and C-terminal regions. Competition experiments using the 5' and 3' arms of either the vRNA or cRNA panhandle indicated that the N-terminal binding site is shared by both templates. The data indicate that the PB1 RNA-binding sites are constituted by: (i) residues located at the N-terminus (probably common for vRNA and cRNA binding) and, either (ii) residues from the central part of PB1 (for cRNA) or (iii) residues from the C-terminal region of PB1 (for vRNA), and suggest that PB1 undergoes a conformational change upon binding to cRNA versus vRNA templates.
In situ hybridization of mouse satellite complementary RNA to the chromosomes of the mouse ascites tumor line MSWBS revealed that the distribution of satellite DNA sequences paralleled the location of constitutive heterochromatin as defined by the C-banding technique.
Electron microscopic examination of hepatoma tissue culture (HTC) cells revealed low numbers of intracisternal type A particles (IAP) and type C viruses. Exposure of HTC cells to either 10(-4) or 10(-5) M5-bromo-2'-deoxyuridine (BUdR) caused a more than 50-fold increase in the number of IAP observed with the electron microscope. The number of IAP increased after only 2 days of growth in 10(-5) M BUdR, whereas 3 days of growth in 10(-4) m BUdR were necessary to observe an increase. A 2-day pulse of 10(-4) M BUdR was also sufficient to cause the increase in type A particles, provided the cells were continued in culture for another 2 days. Unlike many other cell lines, HTC cells treated with BUdR did not show an increase in type C viruses. This conclusion was based on the observations that BUdR treatment caused no detectable increase in extracellular particulate viral reverse transcriptase or in viral RNA complementary to a DNA probe made to a rat endogenous type C virus (RaLV).
The nucleotide sequence of the transcript of the "late" strand of the region of SV40 DNA preceding the preferred initiation site for Escherichia coli RNA polymerase has been determined to be U-G-U-A-A-C-C-A-U-U-A-U-A-A-G-C-U-G-C-A-A-U-A-A-A-C-A-A-G-U-U-A-A-C-A-A-C-A-A-C-A-A-U-U-G-Cp. Hemophilus influenza restriction endonuclease cleaves this region 30 nucleotides (base pairs) before the site of initiation of RNA synthesis by RNA polymerase.
The nucleotide sequence of the RNA transcript from the "early" (E) strand of SV40 DNA immediately preceding the preferred E. coli RNA polymerase start site is G-(A-A-A-C, -A-U-)-A-A-A-A-U-G-A-A-U-G-C-A-A-U-U-G-U-U-G-U-U-G-U-U-A-A-C-U-U-G-U-U-U-A-U-U-G-C-A-G-C-U-U-A-U-A-A-U-G-G-U-U-A-C-Ap. The last nucleotide of the sequence is the first nucleotide transcribed by E. coli RNA polymerase from the "E" strand. The DNA template contains a palindrome of 17 residues that includes the Hemophilus influenza restriction endonuclease cleavage site G-T-T-A-A-Cp. The DNA which gives this transcript lies very close to one end of SV40 DNA segment in the Adeno-SV40 hybrid virus Ad2+ND3 and appears to contain sufficient untranscribed information to specify the E. coli RNA polymerase start.
We describe the intron-exon structure of and the homology among the four alpha-tubulin genes of Drosophila melanogaster. Three of the genes share a highly conserved 1.3 kb sequence which corresponds to most of the RNA complementary portion of the genes. The fourth gene is different. Its 5' half has weak homology and its 3' half has moderate homology to the other three genes. The homology maps were first determined by electron microscopy of heteroduplexes between pairs of genes. Higher resolution maps were then obtained by gel analysis of heteroduplexes that had been digested with S1 nuclease.
We have determined the sequence of a 1228 base-pair cloned DNA fragment from Bacillus licheniformis capable of specifically complementing mutations in the spoOH gene, which is required for the early stage of sporulation in B. subtilis. The sequence has only one long open reading frame consisting of 168 codons. In vivo and in vitro transcription mapping studies indicate the size of complementary RNA to be around 1 kb with the 5' initiation site at base 79 and the 3' termination site in the area of base 1138. This indicates the presence of a 5' untranslated RNA and a fairly long 3' extension. The promoter sequence of this gene is 5'TATAAT3' at -10, and 5'TTGACG3' at -35, a typical E. coli-like promoter sequence, and is transcribed in vitro specifically only by RNA polymerase containing delta 55 and not delta 37-containing holoenzyme.
In transgenic tobacco, anti-sense control of the chimaeric bar (bialophos resistance) gene is manifested by a reduced bar transcript level and a reduced synthesis of bar translational product (PAT, phosphinothricin acetyl transferase) per transcript (1). This study shows that the anti-bar gene controls the transcript level and the translation efficiency of the bar mRNA through independent events and at different cellular sites. On the basis of these results a model for the mechanism of anti-sense control is proposed.
New synthetic routes have been devised for the high yield preparation of protected 2'-O-allylribonucleoside-3'-O-phosphoramidites, exemplified by the ribonucleosides guanosine and 2,6-diaminopurine riboside (2-aminoadenosine). Key features are the use of versatile intermediates and an easy allylation step. The development of a novel synthon based on 2'-O-allyl-2,6-diaminopurine riboside enables short 2'-O-allyl-oligoribonucleotide probes to be synthesized with adenine replaced by 2-aminoadenine. Thus very stable hybrids with complementary RNA target sequences can be formed due to the formation of the three hydrogen bond 2-amino A.U base pairs.
An in vitro RNA synthesis system mimicking replication of genomic influenza virus RNA was developed with nuclear extracts prepared from influenza virus-infected HeLa cells using exogenously added RNA templates. The RNA synthesizing activity was divided into two complementing fractions, i.e. the ribonucleoprotein (RNP) complexes and the fraction free of RNP, which could be replaced with RNP cores isolated from virions and nuclear extracts from uninfected cells, respectively. When nuclear extracts from uninfected cells were fractionated by phosphocellulose column chromatography, the stimulatory activity for RNA synthesis was further separated into two distinct fractions. One of them, tentatively designated RAF (RNA polymerase activating factor), stimulated RNA synthesis with either RNP cores or RNA polymerase and nucleocapsid protein purified from RNP cores as the enzyme source. In contrast, the other, designated PRF (polymerase regulating factor), functioned as an activator only when RNP cores were used as the enzyme source. Biochemical analyses revealed that PRF facilitates dissociation of RNA polymerase from RNP cores. Of interest is that virus-coded non-structural protein 1 (NS1), which has been thought to be involved in regulation of replication, counteracted PRF function. Roles of cellular factors and viral proteins, NS1 in particular, are discussed in terms of regulation of influenza virus RNA genome replication.
An efficient synthesis of the novel nucleic acid analogs oligodeoxyribonucleotide N3'-->P5' phosphoramidates, where the 3'-oxygen is substituted by a 3'-nitrogen, is described. Synthesis of the title compounds was accomplished by the following synthetic steps. First, 5'-O-DMT base-protected-3'-amino-2',3'-dideoxynucleosides were prepared. The 3'-aminopyrimidines were obtained via the corresponding 2,3'-anhydronucleosides, whereas 3'-aminopurines were derived via 2'-deoxyxylo precursors. Second, using the prepared 3'-aminonucleosides, oligonucleotide N3'-->P5' phosphoramidates were synthesized on a solid support. Oligonucleotide chain assembly was based upon a carbon tetrachloride-driven oxidative coupling of the appropriately protected 3'-aminonucleosides with the 5'-H-phosphonate diester group, resulting in the formation of an internucleoside phosphoramidate link. Fully deprotected oligonucleotide N3'-->P5' phosphoramidates were characterized by ion exchange and reversed phase HPLC, capillary and slab gel electrophoresis and by 31P NMR analysis. Oligonucleotide N3'-->P5' phosphoramidates form remarkably stable duplexes with complementary RNA strands and also with themselves, where the melting temperature of the complexes exceeded that for the parent phosphodiester compounds by 26-33 degrees C. Additionally, duplexes formed by oligonucleotide phosphoramidates with single-stranded DNA were also more thermally stable than those formed by phosphodiesters. The described properties indicate that these compounds may have great potential in oligonucleotide-based diagnostics and therapeutic applications.
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