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Observed frequencies of restriction enzyme cleavage in viral DNAs are compared to those expected from the cleavage site sequence and the known size and nearest neighbour frequencies of the DNAs. It is found that a number of significant deviations from expectation occur, and possible explanations are put forward.

Ciliated protozoa are characterized by generative micronuclei and vegetative polyploid macronuclei. Micronuclei of Stylonychia mytilus contain 1 600 times as much DNA per haploid genome as E. coli. Most of this DNA is shown to be repetitive. The development of the macronucleus involves, as demonstrated by cytology, only 1/3 of the chromosomes which in a first replication phase are polytenized in probably 5 replication steps and appear as giant chromosomes. At this developmental stage considerable amounts of repetitive DNA are still present in the chromosomes. During the subsequent disintegration phase more than 90% of the DNA are eliminated from the macronucleus anlage. The remainder is further replicated five times and composes the final macronucleus. Since this DNA reassociates with a reaction rate almost identical to an ideal second order reaction its kinetic complexity can be determined by comparison with the kinetic complexity of E. coli DNA. Macronuclear DNA reassociates with a kinetic complexity of 26 times the kinetic complexity of E. coli DNA (corrected for GC content) which indicates that macronuclear DNA sequences exist at a ploidy level of 4 096 C. We assume that macronuclear DNA may be present only once per haploid genome. In this case it represents only 1.6% of the DNA in micronuclei or 10% of the DNA in the giant chromosome stage.

The base-pair changes induced by the highly carcinogenic agent, 4-nitroquinoline-1-oxide, have been determined from the reversion rates of defined tester strains and from the amino acid replacements of revertant iso-1-cytochromes c . The mutant codons and the base-pair changes of reverse mutations of 14 cyc 1 mutants were previously determined from alterations of iso-1-cytochromes c in intragenic revertants. These 14 cyc 1 mutants, which were used as tester strains, included nine mutants with altered AUG initiation codons, an ochre (UAA) mutant, an amber (UAG) mutant and three frameshift mutants (Stewart et al. , 1971,1972; Stewart & Sherman, 1972,1974; Sherman & Stewart, 1973). NQO † induced a high rate of reversion in the initiation mutant cyc 1-131, the only mutant in the group which reverts to normal iso-1-cytochrome c by a G · C → A · T transition. In addition, NQO produces a significant rate of reversion of all cyc 1 mutants which revert by G · C transversions, e.g. the amber (UAG) mutant and the initiation mutants containing AGG, and probably CUG mutant codons. It did not revert the ochre mutant which contains no G · C base pairs. Ten NQO-induced revertants of the amber mutant cyc 1-179 contained the expected replacements of residues of tyrosine, and ten NQO-induced revertants of each of the cyc 1-131 and cyc 1-133 initiation mutants all contained the expected normal iso-1-cytochrome c . The structures of these iso-1-cytochromes c and the pattern of reversion of the tester strains indicate that base-pair substitutions arise at G · C base pairs which are the site of NQO attack. Thus NQO induces G · C → A · T transitions, G · C → T · A transversions and possibly G · C → C · G transversions. Because of its mode of action, NQO may be useful in less-defined systems for identifying G · C base pairs in mutant codons.

Current models of the active center of ribosomal peptidyl transferase generally invoke a P-subsite which binds the CCA terminus of peptidyl-tRNA and an Asubsite which binds at least part of the CCA terminus of AA-tRNA [I] . Since the CCA sequence occurs at the 3’-terminus of all tRNAs, it is at least indirectly involved in peptide bond formation. It therefore follows that the elucidation of the role of these sequence in binding to peptidyl transferase sites is a necessary step toward understanding the nature of the peptide bond formation center. One approach which has met with some success in investigating the involvement of the CCA termini of peptidyland AA-tRNAs in the peptidyl transferase reaction has involved the use of N-acylaminoacyl or aminoacyl terminal fragments of tRNA as substrate analogs of the parent molecules [2-l 61. Compounds of this type (e.g., AA-oligonucleotides or AA-nucleosides) can be prepared by chemical synthesis or by enzymatic degradation of the corresponding tRNA derivatives. Despite its complexity, chemical synthesis of substrate analogs for peptidyl transferase offers the important advantage that unnatural compounds, i.e., nonCCA-AA sequences, can be synthesized. The study of such compounds in ribosomal systems can thus enable

It has been shown previously that after self-annealing of the nuclear precursor of messenger RNA (premRNA) at high values of Cot about 20% of the material becomes resistant to ribonuclease digestion [ 1 ] . This was explained by the existence of self-complementary regions in the molecules of pre-mRNA and by the ability of these sequences to form doublestranded complexes after annealing. In this paper we report on the isolation and the most important properties of this fraction of RNA (self-complementary RNA or scRNA). Its possible origin is discussed.

Duplex segments of HeLa-cell nuclear DNA were generated by cleavage with DNA restriction endonuclease from Haemophilus influenzae. About 20–25% of the DNA segments produced, when partly degraded with exonuclease III and annealed, were found to form rings visible in the electron microscope. A further 5% of the DNA segments formed structures that were branched in configuration. Similar structures were generated from HeLa-cell DNA, without prior treatment with restriction endonuclease, when the complementary polynucleotide chains were exposed by exonuclease III action at single-chain nicks. After exposure of an average single-chain length of 1400 nucleotides per terminus at nicks in HeLa-cell DNA by exonuclease III, followed by annealing, the physical length of ring closures was estimated and found to be 0.02–0.1μm, or 50–300 base pairs. An almost identical distribution of lengths was recorded for the regions of complementary base sequence responsible for branch formation. It is proposed that most of the rings and branches are formed from classes of reiterated base sequence with an average length of 180 base pairs arranged intermittenly in HeLa-cell DNA. From the rate of formation of branched structures when HeLa-cell DNA segments were heat-denatured and annealed, it is estimated that the reiterated sequences are in families containing approximately 2400–24000 copies.

When solutions of bacteriophage λ are treated with a water-soluble carbodiimide (CMC), then lysed with formamide and observed with the electron microscope, over 90% of the DNA molecules are found to be attached to phage heads and tails. Sedimentation analysis shows that lysis is complete, and denaturation mapping shows that the right-hand end † of the DNA, and never the left-hand end, is attached to the phage tails. When the carbodiimide-treated phage are lysed and the DNA cleaved with RI endonuclease, the shortest fragment, which contains the right end of λ DNA, is as expected the only fragment found attached to the tails. Comparison of the length of the tail-linked fragment with the length of the free DNA fragment shows that the DNA does not extend to the end of the tail. It does appear to intrude a short distance (30% of the length of the tail, or 130 DNA base-pairs), but this distance is just at the limit of resolution. When phage are placed in 80% formamide and then immediately spread for electron microscopy, about 10% of the phage have partially ejected DNA. Denaturation mapping shows that it is the right end of the DNA which is ejected first.

RNA preparations containing 70-80% mouse κ-chain mRNA have been prepared. The remainder consists of many RNA species, each of which represents a small fraction of the total RNA. The κ-chain mRNA preparation hybridizes with mouse liver DNA with bi-phasic kinetics, indicating that it consists of two fractions —“unique” and “reiterated.” Competition hybridization experiments show that the homology among the unique fractions from different mRNAs is the same as the homology among the amino acid sequences of the corresponding κ-chains. Hence, in addition to the C-region (constant-region) sequences, (most of) the V-region (variable-region) sequences are also derived from unique germ line genes. The reiterated fractions from different κ-chain mRNAs show essentially complete homology with each other. This fraction seems to consist mostly of sequences which do not code for amino-acid sequences of the secreted polypeptide chain, i.e., the “external” section of the mRNA molecule. It is concluded that the number of germ line genes is too small to account for the observed diversity of antibody molecules.

Purified DNA sequences that code for tRNA in Neurospora crassa were isolated and partially characterized. The tRNA cistrons comprise about 0.3 percent of the N. crassa genome. The tRNA:tDNA hybrids showed a buoyant density in cesium sulfate of 1.48 g cm-3 and sedimented in an intermediate position between native DNA and tRNA of N. crassa as expected. Te 50 of hybridized tRNA:tDNA molecules, reassociated tDNA: native DNA and homoduplexes of native DNA were 83.0°C, 88.5°C and 89.5°C respectively from thermal stability studies using hydroxyapatite chromatography. The isolated tRNA cistrons react almost completely with unlabeled DNA and tRNA of N. crassa or tRNA of the slime mutant of N. crassa; but react poorly with either ribosomal RNA or ribosomal RNA cistrons of N. crassa and tRNA of Escherichia coli. It appears that tRNA genes of N. crassa are repeated.

Methoxyamine reacts selectively with tRNA molecules at certain exposed cytosine residues usually located in non base-paired regions of the two dimensional clover leaf structure. Here methoxyamine is used for the first time in a study of a mammalian tRNA structure. One of the sequence abnormalities of myeloma initiator tRNA is a cytosine instead of the usual uracil immediately preceding the anticodon. A study of the reaction of the cytosine residues with methoxyamine indicates that the accessibility of bases to chemical reagents in the anticodon loop of this mammalian initiator tRNA is very similar to that observed for the bacterial initiator tRNA.

The proportion of the Xenopus laevis genome complementary to “7 S” RNA, unfractionated transfer RNA and some selected aminoacyl-tRNAs, and the sequence complexity of these RNA species, have been determined by following the kinetics of RNA-DNA hybridization on filters under conditions of RNA excess at optimum rate temperature. For hybridization of aminoacyl-labelled tRNAs, conditions for optimum aminoacylation were first determined and, where necessary, aminoacyl-tRNAs were treated with nitrous acid to prevent discharge during annealing. Neither the extent nor rate of hybridization was affected by this treatment. “7 S” RNA, coded for by 580 genes per haploid complement of chromosomes, reacts like a single family of nucleotide sequences, whereas about 43 basic tRNA sequences are coded for by at least 7800 genes. If hybrids are not treated with RNase A, the apparent tDNA redundancy is some 23% greater but no more nucleotide sequences are detectable. Taken together, the results suggest that each tRNA sequence is, on average, 200-fold reiterated. The reiteration varies, however, for different aminoacyl-tRNAs. Thus, hybridization resolves only one valyl-tRNA which is coded for by 240 genes, but at least four leucyl-tRNA sequences can be distinguished by hybridization, each of which is on average 90-fold reiterated. Reiteration also varies for the two methionyl-tRNAs detectable both by hybridization and by reversed phase chromatography: tRNA1Met and tRNA2Met are 310- and 170-fold reiterated, respectively, and each is kinetically homogeneous. These saturation values are almost exactly additive and are not influenced by the presence of other tRNA species. Thus the results suggest that Xenopus tRNAs are no more heterogeneous than would be predicted by the genetic code, despite the high but variable multiplicity of tRNA cistrons.

Messenger RNA was prepared from developing sea urchin gastrulae by puromycin release from polyribosomes. Approximately 60% of the total mRNA radioactivity of the postnuclear supernatant was recovered and shown to be free of any other labeled RNA species such as ribosomal and nuclear RNA. The mRNA was examined by hybridization to DNA present in great excess. The mRNA hybridizes almost exclusively with nonrepetitive DNA. Almost all of the messenger RNA molecules of sea urchin gastrulae therefore consist of transcripts from nonrepetitive sequences. It appears that the structural genes expressed at this stage are typically not repeated in the genome and the mRNA does not include recognizable repetitive sequence.

DNA from fully repressed chicken erythrocyte nuclei was examined for stably associated RNA which could possibly serve a role in genetic repression. Alkaline hydrolysates of DNA purified on cesium chloride gradients showed an absence of sufficient ribonucleotides, in stable association with DNA, on both thin layer chromatography and by in vitro tritium labeling to serve to localize or identify all the deoxynucleotide gene sequences in the fully repressed chicken erythrocyte nucleus.

In vivo , ribosomal RNA of Saccharomyces cerevisiae is transcribed from the light strand of γ DNA. In vitro , γ DNA is transcribed with equal efficiency by both of the yeast nuclear RNA polymerases, polymerase I and polymerase II; however the RNA products synthesized by the two enzymes differ from each other both in size and in the relative composition of guanine and cytosine. RNA synthesized by polymerase I hybridizes preferentially to the light strand, while that synthesized by polymerase II hybridizes equally well to either strand. Selective transcription of the light strand of the ribosomal DNA also occurs when high-molecular-weight total nuclear DNA is used as template.

Over 100 regulatory mutations linked to the histidine (his) operon of S. typhimurium have been isolated. They all map in a region estimated to be several hundred base pairs in length located at one end of the his operon ("the hisO region"). The mutations are located at sixteen recombinationally separable sites or are deletions encompassing several sites. Data obtained from pairs of reciprocal three-point tests show that "constitutive" (high enzyme levels) and "promoter-like" (low enzyme levels) hisO mutations are interspersed on the genetic map. In a few crosses, recombination was not observed to occur between markers shown to occupy different sites based on behavior in other recombination tests.

We have studied the viral-specific RNA synthesized after infection of a permissive cell line with Kilham rat virus (KRV). The RNA was shown to be virus specific by analysis of its nucleotide base ratios and by hybridization with KRV and cellular DNA. Viral RNA is synthesized as early as 2 h after infection. This viral RNA synthesis occurs before viral progeny DNA synthesis which is initiated at 7 to 8 h after infection. The predominant viral RNA synthesized before and after viral progeny DNA synthesis has a sedimentation coefficient of approximately 18 S in dimethylsulfoxide-sucrose gradients and a calculated molecular weight of 6.5 × 10 5 to 7.5 × 10 5 . KRV contains a molecule of single-stranded DNA with a molecular weight of approximately 1.6 × 10 6 . If the viral-specific 18 S RNA is a homogenous species, it would account for 40 to 50% of the viral genome. A small amount of 26 S viral RNA with a molecular weight of 1.6 × 10 6 to 1.7 × 10 6 can also be detected. If this 26 S RNA is a single viral-specific entity, it could represent a transcription of the entire KRV genome.

Digestion of Rous sarcoma virus 32 P-labeled high molecular-weight RNA with T 1 ribonuclease resulted in the release of approximately 30 unique large oligonucleotides which were resolved by electrophoresis and chromatography on thin layers of cellulose and DEAE-cellulose. Determination of the radioactivity present in each oligonucleotide of known chain length and in the remaining digestion products made it possible to estimate the total number of residues expected in a polynucleotide chain containing the sequences represented. The oligonucleotides are present in amounts greater than would be expected for a unique and nonrepetitive 70 S RNA molecule with a molecular weight of 1 × 10 7 . Many of the sequences must, therefore, be present in more than one of the 35 S RNA subunits comprising the 70 S RNA.

Summary Large T 1 RNase fragments of 18S and 28S rRNA and their nucleolar precursors were separated by successive elution from columns of DEAE Sephadex at pH 7.6 and DEAE-cellulose at pH 3.4, respectively. The fragments that contain the methylated oligonucleotides Am-Gm-Cm-Ap, Um-Gm-Up, Gm-Up and Cm-Ap are referred to as α2 −2 , α2 −4 , α2 −3 and α1 −1 ′. Fragment α2 −2 is the eicosanucleotide Am-Gm-Cm-A-A-A-U-U-C-A-U-A-U-U-C-A-A-A-C-Gp which is the 5′ end of 28S rRNA and its precursors including nucleolar 45S RNA. The dinucleotide Cm-Ap is present only in 18S rRNA and 45S nucleolar RNA.

A double mutant A1G82 of the su III + tyrosine gene in Escherichia coli was constructed by genetic cross. This mutant is a stronger glutamine-inserting amber suppressor than either of the single mutants A1 or G82.

To determine whether bacterial ribosomes recognize a distinguishing feature in the immediate vicinity of actual initiator codons or are directed to these sites through involvement of other portion(s) of the mRNA molecule, the interaction between ribosomes and defined 32 P-labeled initiator fragments from R17 RNA was studied. When incubated with mixtures of the three sites, ribosomes from Bacillus stearothermophilus (which initiate only the A protein on intact phage RNA) are able to select out the A fragment and discriminate against the coat and replicase initiator regions. By contrast, Escherichia coli ribosomes do not rebind that coat-protein region of R17 most efficiently, as they in the native RNA, but likewise prefer the A-protein initiator fragment. In both cases, ribosome binding of the isolated A site is comparable by several criteria to normal polypeptide-chain initiation on an intact R17 messenger RNA in vitro . E. coli ribosomal preference for the A site is confirmed in experiments with randomly fragmented R17 RNA, by both the initiation dipeptide and ribosome protection assay. Thus the A-protein ribosome-binding site of R17 RNA appears intrinsically to be a good initiator, while efficient recognition of the coat and replicase regions requires the participation of some portion of the remainder of the phage RNA molecule.

70-S initiation complexes of Escherichia coli ribosomes and 32P-labelled Qβ RNA were digested with T1 ribonuclease at 0°C, and the ribosome-protected Qβ initiator fragments resolved by poly-acrylamide gel electrophoresis. The sequence of one of the fragments (83 nucleotides long) has been determined. It begins with the sequence of 32 nucleotides already determined for the ribosome binding site of the replicase cistron. The sequence of the remaining 51 nucleotides establishes the order of a further 17 amino acids in the phage-coded replicase subunit. Seven out of the first 11 amino acids in the replicase subunits of phages MS2 and Qβ are identical.

The relationship of biologic activity and polyadenylic acid (poly(A)) content of human reticulocyte messenger RNA has been investigated. Purified 6 to 16 S RNA was fractionated on an oligodeoxythymidylate-cellulose column and assayed for (a) messenger RNA content by hybridization to [3H]CTP-labeled complementary DNA, (b) biologic activity in a Krebs ascites cell-free system, and (c) poly(A) content by hybridization to [3H]polyuridylate. The results indicate that biologic activity of human reticulocyte messenger RNA is independent of its poly(A) length and proportional to its messenger RNA content.

Nalidixic acid can efficiently induce the reversion of some T4 rII mutations. The great majority of the strains whose reversion can be induced by this antibiotic are also sensitive to the mutagenic action of proflavin, indicating that mutagenicity of nalidixic acid results in base pair addition or deletion. With bacterial host strains resistant to nalidixic acid, the mutagenic effect is greatly reduced but not the effects on phage multiplication. This fact shows that the mutagenic agent is not the nalidixic acid itself but a derivative synthesized in sensitive bacterial strains.

Eco R 1 restriction endonuclease makes two cuts in each repeating unit of amplified ribosomal DNA (rDNA) from Xenopus laevis . The locations of these cuts have been established by comparison of the secondary structure of single-stranded Eco R 1 fragments (as visualized in the electron microscope) with that of X. laevis rRNAs and of long strands of uncut rDNA. Of the two classes of fragments generated, the smaller one contains 90% of the 28S rRNA gene, has a duplex molecular weight of 3.0 × 10 6 and is homogeneous in size. The larger class of molecules contains 80% of the 18S rRNA gene and all of the nontranscribed spacer. These latter fragments are heterogeneous with molecular weights ranging from 4.0 to 5.9 × 10 6 . The distribution of sizes within the large class of fragments varies among different preparations of rDNA, and heterogeneity is present in the DNA amplified from the rDNA of a single nucleolar organizer. The heterogeneity is located in the nontranscribed spacer region which is variable in length. This has been demonstrated by the formation of deletion loops in heteroduplexes made between larger fragments of different lengths.

The female-specific coliphages T7, T3, φI, φII, W31, and phage H have been compared by several biochemical techniques. The DNA base-sequence homologies have been determined by examining the appropriate heteroduplexes in the electron microscope. The patterns of RNA and protein syntheses have been investigated by electrophoreses of the radioactively labeled material on polyacrylamide gels. Phages φI, φII, W31, and H are clearly closely related to T7 and more distantly related to T3.

An enzymatic activity that terminally cross-links T7 DNA has been detected in extracts from phage T7-infected Escherichia coli cells. This activity required the functional products of gene 1.3 (T7 DNA ligase), gene 5 (T7 DNA polymerase), and gene 6 (T7 exonuclease) as well as Mg2+ ions, the four deoxytriphosphates, and ATP. The cross-linking system could be reconstituted with purified ligase, polymerase, and exonuclease, and maximal activity required the presence of all three enzymes. The system was active on phage DNA which possessed unique termini but not on DNA with non-unique ends.

Messenger RNA of the cellular slime mold, Dictyostelium discoideum , contains about equimolar amounts of two classes of poly(adenylic acid) [poly(A)]; one is about 25 nucleotides long and the second about 100 nucleotides long. At least half of the messenger RNA molecules contain one sequence each of poly(A) 25 and poly(A) 100 ; both poly(A) sequences are located near the 3′ end of messenger RNA, and the kinetics of their appearance on messenger RNA precursor indicates that poly(A) 25 is added before poly(A) 100 . Dictyostelium nuclear DNA contains 14,000-15,000 sequences of poly(dT) 25 which could code for the smaller poly(A) residues. The poly(A) 100 must be added post-transcriptionally. The poly(dT) 25 sequences are interspersed throughout the genome and may well represent transcription termination regions.

Summary Evidence for intramolecular base composition heterogeneity in kinetoplast DNA has been found by high resolution melting analysis. The specific melting profiles, obtained for the kinetoplast DNA isolated from four different species, indicate that extensive variations in base sequence occurred in this DNA during evolution of the trypanosomatidae.

Analysis of the interaction between mRNA codons and tRNA anticodons suggests a model for the evolution of the genetic code. Modification of the nucleic acid following the anticodon is at present essential in both eukaryotes and prokaryotes to ensure fidelity of translation of codons starting with A, and the amino acids which could be coded for before the evolution of the modifying enzymes can be deduced.

A method is described which allows estimates to be made of the purity or identity of preparations of purified mRNA species.

Pyrimidine oligodeoxyribonucleotides, comprising those sequences of pyrimidine nucleotides that are flanked by a purine nucleotide at each end in the original nucleic acid strand, were released from f1 DNA by the action of diphenylamine in acid solution and separated by ion exchange chromatography. Improvements of earlier analytical techniques have permitted the assembly of a complete catalog of frequencies of occurrence of these sequences. All possible components of different base composition up to chain length 4 were detected, but some of the possible cytosine-rich sequences are missing from the groups of chain lengths 5–8. No sequence of consecutive thymine deoxyribonucleotides of chain length greater than 5 was detected. Ten sequences of chain lengths ranging from 6 to 19 were found to occur only once in each DNA molecule, the last representing the longest sequence of consecutive pyrimidine deoxyribonucleotides yet found in DNA of any origin. The unique sequences of chain lengths 13 and 19 are rich in cytosine and would not be expected to occur at all in a randomly arranged polymer with the base composition of this DNA.

1. The sites within the tRNA sequence of nucleosides methylated by the action of enzymes from mouse colon, rat kidney and tumours of these tissues acting on tRNA(Asp) from yeast and on tRNA(Glu) (2), tRNA(fMet) and tRNA(Val) (1) from Escherichia coli were determined. 2. The same sites in a particular tRNA were methylated by all of these extracts. Thus tRNA(Glu) (2) was methylated at the cytidine residue at position 48 and the adenosine residue at position 58 from the 5'-end of the molecule; tRNA(Asp) was methylated at the guanosine residue at position 26 from the 5'-end of the molecule; tRNA(fMet) was methylated at the guanosine residues 9 and 27, the cytidine residue 49 and the adenosine residue 59 from the 5'-end; tRNA(Val) (1) was methylated at the guanosine residue 10, the cytidine residue 48 and the adenosine residue 58 from the 5'-end. 3. All of these sites within the clover leaf structure of the tRNA sequence are occupied by a methylated nucleoside in some tRNA species of known sequence. It is concluded that methylation of tRNA from micro-organisms by enzymes from mammalian tissues in vitro probably does accurately represent the specificity of these enzymes in vivo. However, there was no evidence that the tumour extracts, which had considerably greater tRNA methylase activity than the normal tissues, had methylases with altered specificity capable of methylating sites not methylated in the normal tissues.

The complexity of Rous sarcoma virus RNA has been determined using molecular hybridization. Relative to poliovirus RNA, the complexity of Rous sarcoma virus is 9·3 × 10 6 daltons, a value close to its physically-determined molecular weight of about 10 7 . Our interpretation is that the 35 S RNA subunits of the 70 S virus genome are non-repetitive, that is, each possesses a unique nucleotide sequence, although a limited amount of redundancy cannot be excluded.

Data on DNA base composition heterogeneity and molecular size, mainly from bacteria, are compiled from the literature. A series of theoretical models of increasing complexity is put forward, and it is concluded that the data support a model in which the DNA consists of a sequence of “segments” with different underlying base compositions. Estimates of mean segment length and variance of G + C content are made. The possibility that the excess heterogeneity above random results from variation in the composition of coded proteins is considered and rejected. It is concluded that the heterogeneity is probably caused by variations in the relative use of synonymous codons in different genes.

The constitutive heterochromatin, as demonstrated by the C band technique, may be subdivided into a number of categories when other characteristics are considered. The responses to fluorochromes QM and 33258 Hoechst, the behavior following G band staining, the repetitive DNA content, and many other criteria are useful for the classification of heterochromatin. The heterochromatin patterns of three mammalian species are presented to demonstrate that within each karyotype there may be several different types of C bands. In general, a correlation may also be made between GC-rich satellite DNA and dull (or negative) Q fluorescence, and between AT-rich satellite DNA and bright Q, fluorescence.

Earlier we described a method for the purification of the transcribing strand of Salmonella typhimurium rDNA. Here we report the purification of the complementary (minus) strand of Escherichia coli and Salmonella typhimuriunz rDNA, by means of partial reassociation of total bacterial DNA, chromatography on hydroxyapatite, on methylalbumin-kieselguhr, and again on hydroxyapatite. A method is presented for the measurement of rDNA(minus). From the purified complementary strands of rDNA a stable duplex has been reconstructed with nearly 50°/, rDNA content. DNA sequences coding for ribosomal RNA occur probably in six copies in the chromosome of Escherichia coli, comprising 0.3-0.4°/0 of the DNA [l]. The same is true for Salmonella typhimurium. Several procedures have been published for the purification of the transcribing (further : plus) strand of this rDNA in the form of rRNA-DNA hybrids from Bacillus subtilis, E. coli and S. typhimurium [2-51, but no successful purification of the double-stranded bacterial rDNA has been achieved. Here we report the partial purification of the non-transcribing (further : minus) strand of bacterial rDNA, a modified version of our earlier procedure for the purification of the plus strand [5], the reconstruction of a double-stranded structure from the complementary strands and characterization of the resulting rDNA duplex.

The total nucleotide sequence of tRNAGlu3 from baker's yeast was determined. This tRNA specifically recognizes the GAA but not the GAG codon for glutamic acid. The existence of a minor nucleoside, 2-thiouridine-5-acetic acid methyl ester (S), was confirmed at the “Wobble position” in the anticodon of this tRNA. The specificity of the coding properties of this tRNA can be explained in terms of the structure and position of this minor base.