Subnanomole detection and quantitation of high specific activity 32P-nucleotides.

Microbore liquid chromatography utilizes conventional HPLC and ultraviolet detection principles to determine subnanomole mass quantities of biologically significant molecules. This system takes advantage of specifically designed microflow equipment to analyze ultraviolet absorbing species at the picomole range. 32P-labeled nucleotides are examples of compounds routinely used at picomole quantities that are extremely difficult to accurately quantify using standard mass measurement techniques. The procedure described in this paper has the capability of measuring nucleotides down to 10 pmol using commercially available microbore ultraviolet detection equipment. The technique can be used to accurately measure the specific activity of as little as 10 microCi of an aqueous 32P-nucleotide solution.

Use of 33P for Sanger DNA sequencing.

The use of [alpha-33P]deoxyadenosine 5'-triphosphate ([alpha-33P]dATP) in DNA sequencing has been described. 33P has a maximum beta-emission energy that is 50% stronger than 35S, but fivefold weaker than 32P. As a result, sequences generated using [alpha-33P]dATP have short exposure times like 32P, yet they maintain band resolution similar to 35S. Handling of [alpha-33P]dATP is straightforward because no special lead or Plexiglas shielding is necessary.

The gene for a spinach chloroplast isoleucine tRNA has a methionine anticodon.

The nucleotide sequence of the gene for spinach chloroplast tRNAIle1 has been determined. The gene is found in two copies located in the inverted repeat regions of spinach chloroplast DNA, but not within the ribosomal RNA spacer. Both copies of the tRNAIle1 gene have been sequenced and found to be identical. A very unusual characteristic of the tRNAIle1 gene is that the anticodon is CAT which is a methionine anticodon. In the tRNA the C residue in the anticodon is subsequently modified, presumably to prevent misreading of the genetic code. The spinach chloroplast tRNAIle1 gene is colinear with its RNA sequence and does not contain an intervening sequence as has been reported for maize chloroplast tRNAIle2 (Koch, W., Edwards, K., and Kossel, H. (1981) Cell 25, 203-213). The tRNAIle1 gene does not code for the 3'-terminal CCA end, nor do any other tRNA genes appear to be contiguous with this gene.

Structure of a spinach chloroplast threonine tRNA gene.

The gene for spinach chloroplast tRNAThr3 has been sequenced and is co-linear with the tRNA, does not contain an intervening sequence, and does not code for the 3'-terminal CCA, which is added post-transcriptionally. This gene shares features with prokaryotic, eukaryotic, and mitochondrial tRNA genes. The opposite strand of the tRNA gene contains a proper ribosome binding site, homology to a classical bacterial promoter, and could potentially code for a small peptide.

Nucleotide sequence of a spinach chloroplast valine tRNA.

The nucleotide sequence of a spinach chloroplast valine tRNA (sp. chl. tRNA Val) has been determined. This tRNA shows essentially equal homology to prokaryotic valine tRNAs (58-65% homology) and to the mitochondrial valine tRNAs of lower eukaryotes (yeast and N. crassa, 61-62% homology). Sp. chl. tRNA Val shows distinctly lower homology to mouse mitochondrial valine tRNA (53% homology) and to eukaryotic cytoplasmic valine tRNAs (47-53% homology). Sp. chl. tRNA Val, like all other chloroplast tRNAs sequenced, contains a methylated GG sequence in the dihydrouridine loop and lacks unusual structural features which have been found in several mitochondrial tRNAs.

The nucleotide sequence of a major species of leucine tRNA from bovine liver.

Through the use of a variety of post-labeling techniques, the nucleotide sequence of a major species of leucine tRNA from bovine liver was determined to be pG-G-U-A-G-C-G-U-G-m-G-C-ac-C-G-A-G-C-G-G-D-C-psi-A-A-G-G-C-m-G-C-U-G-G-A-psim- U-I-A-G-m-G-C-psi-C-C-A-G-U-C-psi-C-psi-U-C-G-G-G-G-G-m-C-G-U-G-G-G-T-psi-C-G-m -A-A-U-C-C-C-A-C-C-G-C-U-G-C-C-A-C-C-AOH. A comparison of known sequences of leucine tRNAs shows a consistent set of features which clearly distinguish prokaryotic and eukaryotic leucine tRNAs from each other.

The nucleotide sequence of spinach chloroplast methionine elongator tRNA.

The nucleotide sequence of spinach chloroplast methionine elongator tRNA (sp. chl. tRNAm Met) has been determined. This tRNA is considerably more homologous to E. coli tRNAm Met (67% homology) than to the three known eukaryotic tRNAm Met (50-55% homology). Sp. chl. tRNAm Met, like the eight other chloroplast tRNAs sequenced, contains a methylated GG sequence in the dihydrouridine loop and lacks unusual structural features which have been found in several mitochondrial tRNAs.

The nucleotide sequence of spinach cytoplasmic 5 S ribosomal RNA.

The nucleotide sequence of the cytoplasmic 5 S ribosomal RNA from Spinacia oleracea has been determined. A secondary structural model possessing four base-paired regions can be constructed from the primary structure. This RNA shows 90 to 93% nucleotide sequence homology with other higher plant cytoplasmic 5 S RNAs and 73% homology with that of the lower eukaryote Chlorella. The spinach 5 S RNA has the nucleotide sequence identical with that of Chlorella in two important single-stranded regions, the sequence C10 AUACC and the dodecanucleotide sequence at positions 33 to 44. A nucleotide sequence similar or identical with C10 AUACC is found in most other eukaryotic 5 S RNAs, including the 5 S RNA from human KB cells. In addition, a single-stranded loop of 12 residues corresponding to positions 33 to 44 in the spinach 5 S RNA sequence may be a general feature of eukaryotic cytoplasmic 5 S RNAs, while prokaryotic 5 S RNAs have a 13-member loop for the corresponding residues. Several other important homologies in primary and secondary structure have also been observed in comparing spinach 5 S RNA to other 5 S RNAs.

Nucleotide sequence of a spinach chloroplast proline tRNA.

The nucleotide sequence of a spinach chloroplast proline tRNA (sp. chl. tRNApro) has been determined. This tRNA shows more overall homology to phage T4 proline tRNA (61% homology) than to eukaryotic proline tRNAs (53% homology) or mitochondrial proline tRNAs (36-49% homology). Sp. chl. tRNApro, like all other chloroplast tRNAs sequenced, contains a methylated GG sequence in the dihyrouridine loop and lacks unusual structural features which have been found in many mitochondrial tRNAs.

Primary structure of bovine pituitary secretory protein I (chromogranin A) deduced from the cDNA sequence.

Secretory protein I (SP-I), also referred to as chromogranin A, is an acidic glycoprotein that has been found in every tissue of endocrine and neuroendocrine origin examined but never in exocrine or epithelial cells. Its co-storage and co-secretion with peptide hormones and neurotransmitters suggest that it has an important endocrine or secretory function. We have isolated cDNA clones from a bovine pituitary lambda gt11 expression library using an antiserum to parathyroid SP-I. The largest clone (SP4B) (approximately equal to 1.6 kilobases) hybridized to a transcript of 2.1 kilobases in RNA from parathyroid, pituitary, and adrenal medulla. Immunoblots of bacterial lysates derived from SP4B lysogens demonstrated specific antibody binding to an SP4B/beta-galactosidase fusion protein (160 kDa) with a cDNA-derived component of 46 kDa. Radioimmunoassay of the bacterial lysates with SP-I antiserum yielded parallel displacement curves of 125I-labeled SP-I by the SP4B lysate and authentic SP-I. SP4B contains a cDNA of 1614 nucleotides that encodes a 449-amino acid protein (calculated mass, 50 kDa). The nucleotide sequences of the pituitary SP-I cDNA and adrenal medullary SP-I cDNAs are nearly identical. Analysis of genomic DNA suggests that pituitary, adrenal, and parathyroid SP-I are products of the same gene.

Homology between chloroplast and prokaryotic initiator tRNA. Nucleotide sequence of spinach chloroplast methionine initiator tRNA.

The nucleotide sequence of a chloroplast methionine initiator tRNA from spinach has been determined. Although from a eukaryotic organism, this tRNA strongly resembles prokaryotic initiator tRNAs. Spinach chloroplast tRNAMetf has a much higher sequence homology with prokaryotic initiator tRNAs (81 to 84%) than with eukaryotic initiator tRNAs (64 to 69%). In addition, it possesses the two unique features of prokaryotic initiator tRNAs, lacking a base pair between the 5'-terminal residue and the fifth nucleotide from the 3'-end and containing a T-psi-C-A sequence in loop IV. Also, like prokaryotic initiator tRNAs, the chloroplast tRNAMetf is 77 nucleotides long and has few modified nucleosides (2'-O-methylguanosine, dihydrouridine, 7-methylguanosine, ribothymidine, and pseudouridine). This chloroplast initiator tRNA is strikingly different in sequence homology (55 to 62%), number of residues, and structure from mitochondrial initiator tRNAs. Restriction enzyme mapping techniques have shown that the chloroplast tRNAMEtf hybridizes to spinach chloroplast DNA. A set of characteristic chloroplast tRNA features seems to be emerging from a comparison of this tRNAMetf and several other chloroplast tRNAs which have been completely or partially sequenced. All have a 2'-O-methylated G-G sequence in the dihydrouridine loop, and the sequence T-psi-C-A, as opposed to T-psi-C-G, is predominantly found in loop IV. This is the reverse of the situation encountered in the overall non-chloroplast tRNA population.

Nucleotide sequence of a spinach chloroplast threonine tRNA.

The nucleotide sequence of a spinach chloroplast threonine tRNA has been determined. This chloroplast threonine tRNA has been determined. This chloroplast threonine tRNA has 75 nucleotides, which is the same chain length as the recently determined threonine tRNA from yeast mitochondria. This contrasts with the 6 non-organelle threonine tRNAs sequenced to date, which are 76 nucleotides in length. However, other than this similarity in size, the chloropast tRNAThr has little similarlity to the yeast mitochondrial threonine tRNA, and shows essentially equal homology to both prokaryotic and eukaryotic feature of sequence homology to other threonine tRNAs, there is a 19-nucleotide segment encompassing the entire T psi C stem and loop region, that, except for post-transcriptional modifications, is identical in this spinach chloroplast tRNAThr and in yeast cytoplasmic tRNAThr1A. A most unusual feature of this spinach chloroplast tRNAThr is that it has an A residue at the 5'-end of the anticodon loop, a site that is occupied by a pyrimidine in all other tRNAs sequenced to date. Other than this feature, the tRNAThr contains all of the invariant and semi-invariant residues normally found in tRNAs. This tRNA hybridizes to spinach chloroplast DNA, but does not hybridize to the same region of the spinach chloroplast genome that contains the genes for spinach chloroplast tRNA1Thr or tRNA2Thr. This tRNA therefore appears to be a third isoaccepting species of threonine tRNA encoded by spinach chloroplast DNA.