A technique for mapping transfer RNA genes by electron microscopy of hybrids of ferritin-labeled transfer RNA and DNA: the phi-80hpsu+3-system

A method for mapping transfer RNA genes on single strands of DNA is described. tRNA is covalently coupled to the electron-opaque label, ferritin. The ferritinlabeled tRNA, Fer-tRNA, is hybridized to a single strand of DNA, or to a single- strand region of a DNA in a heteroduplex. The sites where the Fer-RNA binds to the complementary sequence on the DNA are then mapped by electron microscopy. Several alternative coupling procedures are described (see Fig. 1). In HzI a — COCH₂Br group is attached to ferritin by acylation. 3'-Oxidized tRNA is joined to HSRCONHNH₂ by hydrazone formation. Ferritin is then coupled to tRNA by reaction of the CBr and SH bonds. In the BI procedure a lysine amino group of ferritin is coupled by Schiff base formation with 3'-oxidized RNA. The conjugate is stabilized by borohydride reduction. In the BII procedure, a —COCH₂Br group is attached to ferritin. (H₂NCH₂CH₂S—)₂ is coupled to oxidized tRNA by Schiff base formation and borohydride reduction. An SH group is exposed by reduction. This HS-tRNA is coupled to a —COCH₂Br group attached to ferritin. All the procedures work but BII is recommended. Methods for purifying the Fer-tRNA and the Fer-tRNA-DNA hybrid are described. For the transducing phages, φ80hpsu^+,?_III and φ80hpsu^?_III, the DNA molecules each carry a piece of bacterial DNA of length 0·066±0·007 λ unit (3100 nucleotide pairs; we find the length of λ is 8·99 φX174 units) replacing a piece of phage DNA of φ80h of length 0·045±0·005 λ unit. The left junction of this bacterial DNA with phage DNA (referred to as P-B′) is at or close to the att site. The two tandem tyrosine genes of φ80hpsu^+,?_III and the single tRNA gene of φ80hpsu^?_III have been mapped at a position 1100 nucleotides to the right of the left (P·B′) junction of phage DNA and bacterial DNA, by hybridizing Escherichia coli Fer-tRNA to φ80hpsu_III/φ80h heteroduplexes. The separation of the two ferritin labels in φ80hpsu^+,?_III hybrids gives 140±20 nucleotides as the size of a single tyrosine tRNA gene.     

An mRNA-protein fusion at N-terminus for evolutionary protein engineering

A novel method to link a nascent protein (phenotype) to its mRNA (genotype) covalently through the N-terminus was developed. The mRNA harboring amber stop codon at just downstream of initiation site was hybridized with hydrazide-modified ssDNA at upstream of coding region and was ligated to the DNA. This construct was then modified with 4-acetyl-phenylalanyl amber suppressor tRNA. This modified construct was fused with the nascent protein via the phenylalanine derivative when the mRNA uses the amber suppressor tRNA to decode the amber stop codon. The obtained fusion molecule was used successfully in selective enrichment experiments. It will be applicable for high-through-put screening in evolutionary protein engineering. In contrast to fusion molecules generated by other methods in which the protein is linked to genotype molecule through the C- terminus, our fusion molecule will serve to select a protein for which the C-terminus is essential to be active.     

Electron microscopy of late adenovirus type 2 mRNA hybridized to double-stranded viral DNA.

R loops were generated with late adenovirus type 2 (Ad2) mRNA in double-stranded viral DNA, and visualized by electron microscopy. Unpaired DNA sequences in Ad2:Ad2+ND4 heteroduplex DNA served as a visual marker for the orientation of R loops with respect to the conventional DNA map. The most abundant classes of late Ad2 mRNA observed by this technique hybridized, in order of R-loop frequency, with midpoints near posit1ons 0.57, 0.88, 0.77, and 0.40 to 0.50 of the DNA map. The R loop at position 0.57, 0.88, 0.77, and 0.40 containing the hexon gene; the one at position 0.88 corresponded to a region containing the fiber gene. The relative frequencies of these two R loops paralleled those of the encoded gene products. The mRNA sizes, calculated from those of the respective R loops, were slightly larger than needed to code for these polypeptides. Using the R-loop technique, two locations at which adjacent mRNA''s hybridized to different strands were accurately mapped at positions 0.61 and 0.91 of the DNA. The map positions of late Ad2 mRNA correlated well to published RNA and protein maps.     

Chloroplast DNA codes for transfer RNA.

Transfer RNA's were isolated from Euglena gracilis. Chloroplast cistrons for tRNA were quantitated by hybridizing tRNA to ct DNA. Species of tRNA hybridizing to ct DNA were partially purified by hybridization-chromatography. The tRNA's hybridizing to ct DNA and nuclear DNA appear to be different. Total cellular tRNA was hybridized to ct DNA to an equivalent of approximately 25 cistrons. The total cellular tRNA was also separated into 2 fractions by chromatography on dihydroxyboryl substituted amino ethyl cellulose. Fraction I hybridized to both nuclear and ct DNA. Hybridizations to ct DNA indicated approximately 18 cistrons. Fraction II-tRNA hybridized only to ct DNA, saturating at a level of approximately 7 cistrons. The tRNA from isolated chloroplasts hybridized to both chloroplast and nuclear DNA. The level of hybridization to ct DNA indicated approximately 18 cistrons. Fraction II-type tRNA could not be detected in the isolated chloroplasts.     

An electron microscope study of the relative positions of the 4S and ribosomal RNA genes in HeLa cell mitochondrial DNA

The 4S RNA genes in HeLa mitochondrial DNA (mtDNA) have been mapped by electron microscopy using the electron-opaque label ferritin. This method is based on the high affinity interaction between the protein, avidin, and biotin. 4S RNA, covalently coupled to biotin, was hybridized to single-stranded mtDNA. The hybrids were then labeled with ferritin-avidin conjugates. The positions of ferritin-labeled 4S RNA genes were determined relative to the rRNA genes on both heavy (H) and light (L) strands of mtDNA. This region was recognized as a duplex segment after hybridization either with rRNA in the case of H strands or with DNA complementary to rRNA in the case of L strands.Our studies suggest that at least nineteen 4S RNA genes are present in the HeLa mitochondrial genome. On the H strand, we have confirmed the nine map positions found in a previous electron microscope mapping study (Wu et al., 1972) and obtained evidence for three additional 4S RNA genes. On the L strand, seven 4S RNA genes have been mapped. The nineteen genes are distributed more or less uniformly around the genome. There is a pair of closely spaced genes, approximately 150 nucleotides apart, on the H strand, and another closely spaced pair on the L strand.     

Application of the avidin-biotin method of gene enrichment to the isolation of long double-stranded DNA containing specific gene sequences.

A method of enriching for long double-stranded segments of eukaryotic DNA carrying particular genes is described. A purified RNA coded for by the gene is covalently attached to biotin via the protein, cytochrome c. This modified RNA is hybridized to total nuclear, double-stranded DNA under conditions that allow the formation of R-loops. Avidin, which has a high affinity for biotin, is covalently attached to polymer spheres. The complexes of avidin-spheres with DNA:RNA-biotin R-loop hybrids band in CsCl at a much lower bouyant density than does free DNA. This density is a function of the length of DNA coupled per avidin-sphere. This method was used to prepare very long double-strands of DNA highly enriched in the coding sequences for the large rRNAs of D. melanogaster and L. donovani and the histone mRNAs of S. purpuratus.     

Cloning of chicken embryo tRNA genes using single stranded nucleosomal DNA highly enriched for tRNA complementary sequences

DNA from chicken embryo nucleosome tetramers (about 760 base pairs in size) was enriched for tRNA genes by RPC-5 chromatography. The enriched DNA was hybridized with chicken embryo total tRNA and the hybridized DNA isolated utilizing a) avidinbiotin interaction, b) diazobenzyloxymethyl paper, and c) high temperature RPC-5 chromatography. The obtained single stranded DNA highly enriched for tRNA complementary sequences was hybridized with total DNA from nucleosome monomers (140--190 base pairs in size) and the excess of non hybridized monomer nucleosome DNA removed by Sepharose 4B chromatography. The hybrid molecules obtained were made fully double stranded by incubation with E. coli DNA polymerase I, DNA ligase, and exonuclease III. DNA was inserted into plasmid pBR322 by G-C joining procedure and the recombinant DNA used to transform the E. coli strain chi 1776. More than 70% of the transformants obtained hybridize to chicken embryo total tRNA.     

Using a targeted chemical nuclease to elucidate conformational changes in the E. coli 30S ribosomal subunit

Determining the detailed tertiary structure of 16S rRNA within 30S ribosomal subunits remains a challenging problem. The particular structure of the RNA which allows tRNA to effectively interact with the associated mRNA during protein synthesis remains particularly ambiguous. This study utilizes a chemical nuclease, 1, 10-o-phenanthroline-copper, to localize regions of 16S rRNA proximal to the decoding region under conditions in which tRNA does not readily associate with the 30S subunit (inactive conformation), and under conditions which optimize tRNA binding (active conformation). By covalently attaching 1,10-phenanthroline-copper to a DNA oligomer complementary to nucleotides in the decoding region (1396-1403), we have determined that nucleotides 923-929, 1391-1396, and 1190-1192 are within approximately 15 A of the nucleotide base-paired to nucleotide 1403 in inactive subunits, but in active subunits only cleavages (1404-1405) immediately proximal to the 5' end of the hybridized probe remain. These results provide evidence for dynamic movement in the 30S ribosomal subunit, reported for the first time using a targeted chemical nuclease.     

Sequence specific purification of a particular tRNA by solid phase DNA probe.

A novel method for the purification of a specific tRNA using solid phase DNA probe is developed. With this method, the probe DNA immobilized on HPLC gel hybridized with target tRNA within a minute at room temperature. The hybridizing capacity of the solid phase probe was about 20 O.D. per gram dry gel when yeast phenylalanine tRNA was used. The specificity of this method was extremely high and the recovery rate was about 90%.     

An electron microscopic method for the mapping of proteins attached to nucleic acids.

An electron microscopic method for demonstrating the presence of and mapping the positions of proteins specifically bound to nucleic acids is described. The nucleic acid-protein complex is treated with dinitrofluorobenzene under conditions such that dinitrophenyl (DNP) groups are attached to nucleophilic groups on the protein, with only a low level of random attachment to the nuclei acid. This product is treated with rabbit anti-DNP IgG. The position of the protein-(DNP)n(IgG)m complex on the nucleic acid strand can be observed by electron microscopy by protein free spreading methods and, in many cases, by cytochrome-c spreading. If necessary for visualization by the latter method, the size of the labeled region can be increased by treatment with goat anti-rabbit IgG. High efficiency of electron microscopic labeling is achieved. Examples studied are: the adenovirus-2 DNA terminal protein, a protein covalently bound to SV40 DNA, DNA polymerase I bound to DNA, E. coli RNA polymerase bound to T7 DNA, and proteins UV crosslinked to avian sarcoma virus RNA.     

膜上tRNA结合蛋白的分离与初步鉴定

用ＴｒｉｔｏｎＸ－１１４分相法分离啤酒酵母的膜总蛋白,经过酵母ｔＲＮＡ分子交联的Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,用０－０．８ｍｏｌ／Ｌ（ＮＨ４０２ＳＯ４梯度缓冲液洗脱ｔＲＮＡ结合的蛋白质。凝胶阻滞电泳实验室鉴定出两种主要的与ｔＲＮＡ分子特异性结合的蛋白质。     

Secondary structure in transfer RNA genes

The bacterial strand of the heteroduplex of λh80 dglyTsu⁺₃₆tyrTthrT with λh80 carries a cluster of three transfer RNA genes. The bacterial strands of the heteroduplexes of φ80hpsu^+,?_III and φ80hpsu^?_III with φ80h carry two and one genes for tyrosine tRNA, respectively. When these heteroduplexes are spread under weakly denaturing conditions (low formamide), secondary structure features consisting of one or several closely clustered, short duplex regions (folds) are observed. The features map at the positions of the tRNA gene clusters. They are not seen if the DNA is hybridized to Escherichia coli tRNA. It is concluded that the secondary structure features are due to self-complementary sequences in the tRNA genes. In some cases, the duplex folds appear to involve base pairing between sequences on different tRNA genes of a cluster and may also involve the spacer sequences between the tRNA sequences.     

Lupin chloroplast and mitochondrial tRNA populations and their genes

Transfer RNAs isolated from lupin chloroplasts and mitochondria were compared by two-dimensional gel electrophoresis. Twenty chloroplast and 24 mitochondrial tRNA species were identified. The saturation hybridization between lupin chloroplast DNA and 125I-labelled lupin chloroplast tRNAs pointed to the presence of about 34 tRNA genes in lupin chloroplast DNA. The number of mitochondrial tRNA genes estimated by the same method was about 30 genes. EcoRI restriction digest of lupin mitochondrial DNA probed with 32P-labelled lupin mitochondrial tRNAs revealed only a small number of positive restriction fragments. Some of these mitochondrial restriction fragments hybridized with 32P-labelled chloroplast tRNA.     

Peptidyl transferase activity of tRNA: a quantum chemical study

The mechanism of protein synthesis is still unknown due to inability to detect the so-called enzyme "peptidyl transferase" even after elucidation of high-resolution crystal structure of ribosome. We have recently shown by model building and semi-empirical energy calculation that the tRNA molecule at P-site of ribosome may act as peptidyl transferase (Das et al. (1999) J. Theor. Biol. 200, 193-205). We proposed that the tetrahedral intermediate formed from nucleophylic attack of CO of P-site amino-acylated tRNA by NH2 of A-site amino-acylated tRNA is converted to a six-member ring intermediate by conformational change. This ring intermediate produces a free tRNA and a tRNA covalently linked to a peptide. However, energy of the six-member ring intermediate was calculated to be quite high. We show here that the energy values of all the reactants, intermediates and products are within the expected range when they are calculated using high level ab initio quantum chemical methods.     

A new method of in situ hybridization

A new method for gene mapping at the chromosome level using in situ hybridization and scanning electron microscopy is described and has been applied to mapping the rRNA genes of Drosophila melanogaster. Biotin is covalently attached to Drosophila rRNA via a cytochrome c bridge at a ratio of one cytochrome-biotin per 130 nucleotides by a chemical procedure. Polymethacrylate spheres with a diameter of ca. 60 nm are prepared by emulsion polymerization and are covalently attached to the protein avidin at a ratio of 5–20 avidins per sphere. The biotin-labeled rRNA is hybridized to denatured DNA in a chromosome squash. Upon incubation with a sphere solution, some of the biotin sites become labeled with spheres because of the strong non-covalent interaction between biotin and avidin. The chromosome squash is examined in the scanning electron microscope (SEM). Polymer spheres, which are visible in the SEM, are observed to label the nucleolus, where the rRNA genes are located.Contribution number 5121 from the Department of Chemistry.     

Does simian virus 40 DNA integrate into cellular DNA during productive infection?

Late after infection of permissive monkey cells by simian virus 40 (SV40), large amounts of SV40 DNA (30,000 to 220,000 viral genome equivalents per cell) can be isolated with the high-molecular-weight fraction of cellular DNA. Hirai and Defendi (J. Virol.9:705-707, 1972) and H?lzel and Sokol (J. Mol. Biol. 84:423-444, 1974) suggested that this SV40 DNA is covalently integrated into the cellular DNA. However, our data indicate that the high-molecular-weight viral DNA is composed of tandem, "head-to-tail" repeats of SV40 DNA and that very little, if any, of this viral DNA is covalently joined to the cellular DNA. This was deduced from the following experimental findings. The size of the SV40 DNA associated with the high-molecular-weight cellular DNA fraction is greater than 45 kilobases, based on its electrophoretic mobility in agarose gels. In this form the SV40 DNA did not produce heteroduplex structures with a marker viral DNA (an SV40 genome with a characteristic deletion and duplication). After the high-molecular-weight DNA was digested with EcoRI or HpaII endonucleases, enzymes which cleave SV40 DNA once, more than 95% of the SV40 DNA migrated as unit-length linear molecules and, after hybridization with the marker viral DNA, the expected heteroduplex structures were easily detected. Digestion of the high-molecular-weight DNA fraction with restriction endonucleases that cleave cellular, but not SV40. DNA did not alter the electrophoretic mobility of the polymeric SV40 DNA, nor did it give rise to molecules that form heteroduplex structures with the marker viral DNA. Polymeric SV40 DNA molecules produced after coinfection by two physically distinguishable SV40 genomes contain only a single type of genome, suggesting that they arise by replication rather than by recombination. The polymeric form of SV40 DNA is highly infectious for CV-1P monolayers (6.5 X 10(4) PFU per microgram of SV40 DNA), yielding virtually exclusively normal, covalently closed circular, monomer-length DNA. Quite clearly these cells have an efficient mechanism for generating monomeric viral DNA from the SV40 DNA polymers.     

Mammalian prothymosin alpha links to tRNA in Escherichia coli cells.

Prothymosin alpha, a small and highly acidic nuclear protein related to cell proliferation, is known to be covalently attached to a small unidentified cytoplasmic RNA in mammalian cells. Here we demonstrate that recombinant rat prothymosin a links covalently to an RNA when overproduced in Escherichia coli cells. The RNA species of this complex is represented by a wide range of bacterial tRNAs. tRNA(Lys), tRNA(3Ser), tRNA(2Ile), and tRNA(mMet) were identified by sequencing. Prothymosin alpha appears to be linked to the 5' terminus of tRNA. tRNA attachment site lies close to the carboxy-terminus of prothymosin alpha. Furthermore, the carboxy-terminal peptide of prothymosin alpha is also competent for tRNA binding. The site of tRNA attachment coincides with the nuclear localization signal of prothymosin alpha, and tRNA binding might be expected to affect subcellular localization of this protein.     

Expression of the mitochondrial genome in HeLa cells : XIV. The relative positions of the 4 s RNA genes and of the ribosomal RNA genes in mitochondrial DNA

HeLa mitochondrial 4 s RNA has been covalently coupled to the electron opaque label, ferritin, which is visible in the electron microscope. Mixtures of HeLa mitochondrial 12 s ribosomal RNA, 16 s rRNA and/or the 4 s RNA-ferritin conjugate have been hybridized to separated heavy (H) and light (L) strands of HeLa mitochondrial DNA, or to a mixture of H and L strands. The relative positions of the duplex regions corresponding to the 12 s and 16 s rRNA—DNA hybrids and of the ferritin-labeled 4 s RNA's have been mapped in the electron microscope after spreading the DNA strands by the formamide modification of the basic protein film technique. The 12 s and 16 s duplex regions have lengths of 0·-26 ± 0.04 μm and 0.46 ± 0.07 μm, respectively. They are separated by a single-strand region of length 0.047 ± 0.017 μm, corresponding to 160 ± 60 nucleotides. There are nine reproducible binding sites for 4 s RNA on the H strand. One such site lies within the spacer region between the 12 s and 16 s coding sequences, one site is immediately adjacent to the other side of the 12 s sequence and one is adjacent to the other side of the 16 s sequence. The other 4 s sites are rather evenly spaced along the DNA strand of total length 15,600 nucleotides, except that two of them are clustered with a spacing of 120 ± 30 nucleotides between them. There are three 4 s RNA coding sequences on the L strand, separated from one another by 2280 and 3900 nucleotides, respectively.     

Visualization of Z sequences in form V of pBR322 by immuno-electron microscopy.

Form V DNA has been prepared from pBR322 DNA by annealing covalently closed complementary single strands. Specific rabbit antibodies to Z-DNA were shown by radioimmunoassay and electron microscopy to react with form V DNA of pBR322. The bound antibodies were visualized either directly (on synthetic polynucleotides in Z-form), or after reaction with goat anti-rabbit immunoglobulin labeled with ferritin (on form V DNA).