The 3'-terminal nucleotide sequences of lambda DNA

The base sequences of the 3′-termini of coliphage λ DNA have been analyzed by a new technique. Escherichia coli DNA polymerase I was used to add a single radioactive nucleotide to the 3′-OH terminus of one of the DNA strands. The DNA was then digested with pancreatic DNase I, and the resulting oligonucleotides were separated by two dimensional ionophoresis. Terminal oligonucleotides were identified by the presence of the radioactive label, and the base sequence of the labelled terminus was deduced from the base compositions of the terminal di-, tri-, tetra-, etc., oligonucleotides. It is found that the left 3′-terminus of λ DNA ends with the sequence d(pCpGpCpG) and the right 3′-terminus ends with the sequence d(pCpG).     

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.     

Direct determination of DNA nucleotide sequences: structure of a fragment of bacteriophage phiX172 DNA

Methods for the direct determination of nucleotide sequences in DNA have been developed and used to determine the complete primary structure of a fragment of bacteriophage φX174 DNA which is 48 residues in length. This fragment was liberated from φX DNA by digestion at low temperature and high ionic strength with the T4 phage-induced endonuclease IV. The fragment was redigested with endonuclease IV under vigorous conditions and the products fractionated two-dimensionally providing a characteristic endonuclease IV “fingerprint” of the fragment. The Burton (Burton &; Petersen, 1960) depurination reaction was used to characterize the redigestion products and identify the pyrimidine residues at their 5′ and 3′ termini. These oligonucleotide products were then fully sequenced by partial exonuclease digestion with spleen and snake venom phosphodiesterase and analysis of the fractionated digests by base composition, depurination, and 5′ end-group analysis using exonuclease I. Rules for the interpretation of two-dimensional fingerprints of partial exonuclease digests, which rapidly provide sequence information by simple inspection, were also deduced. To derive the complete structure of the fragment, the fully sequenced oligonucleotides were ordered by characterizing large, overlapping, partial endonuclease IV digestion products by means of the depurination reaction. The sequencing methods described are general and may be used for the direct determination of the primary structures of other fragments of DNA.     

Nucleotide sequence analysis of DNA. II. Complete nucleotide sequence of the cohesive ends of bacteriophage lambda DNA

At the ends of bacteriophage λ DNA, the 5′-terminated strands are 12 nucleotides longer than the 3′-terminated strands. The complete sequence of deoxynucleotides in both the protruding 5′-terminated single strands of λ DNA has been determined by partial repair and by complete repair followed by sequencing of isolated oligonucleotides. Starting from the 5′-end of the left-hand cohesive end, the 12 nucleotides are in the sequence dpGpGpGpCpGpGpCpGpApCpCpT. The sequence from the right-hand cohesive end is exactly complementary to that from the left-hand end.     

Nucleotide sequence of a fragment of SV40 DNA that contains the origin of DNA replication and specifies the 5' ends of "early" and "late" viral RNA. III. Construction of the total sequence of EcoRII-G fragment of SV40 DNA.

Limited T1 RNase digestion of subfragments of the SV40 DNA restriction endonuclease fragment EcoRII-G were prepared and analyzed. The fragments were separately labeled with 32P at their 5' terminus and the terminal sequences analyzed with limited snake venom diesterase digestion. The data permitted us to deduce the nucleotide sequence for EcoRII-G. The sequence contains a stretch of 17 A-T base pairs preceding the DNA complementary to the 5' end of "early" message RNA, a stretch of 27 bases with a perfect 2-fold rotational symmetry near the origin of DNA replication and a perfect tandem repeat of 21 nucleotides.     

Nucleotide Sequence Analysis of DNA

There are three major obstacles to the analysis of the nucleotide sequence in a DNA molecule starting from a known location in the DNA molecule. First, it is difficult to obtain large quantities of homogeneous DNA. Second, even the smallest DNA molecules contain several thousand nucleotides which make sequence analysis prohibitive. Third, there are no highly base-specific DNAases available for degrading DNA for sequence analysis. We have overcome some of these obstacles; first, by incorporating highly labelled deoxynucleotides into DNA in vitro, small amounts of material can be used for sequence analysis. Second, the nucleotide sequence of DNA molecules can now be determined from the 5′-terminal. Thus, two dodecanucleotide sequences corresponding to the two cohesive ends of λ DNA have been determined¹ and a nona-decanucleotide sequence corresponding to one cohesive end of phage 186 DNA has been completed². So far, our approach is limited to starting the analysis from the 5′-ends of a DNA molecule. A more general approach is being developed for starting the analysis from other selected parts of a DNA molecule with the use of specifically designed primers.     

Structure of the 3' hairpin termini of four rodent parvovirus genomes: nucleotide sequence homology at origins of DNA replication.

The nucleotide sequences of the 3' termini of the DNA from four autonomous rodent parvoviruses have been determined. The terminus of each genome exists as a Y-shaped hairpin structure involving 115 or 116 nucleotides. The sequence of this region of DNA is highly conserved and shows no evidence of internal sequence heterogeneity, a characteristic which is observed in the terminal nucleotide sequence of the helper-dependent, adeno-associated viruses (Berns et al., 1978a). The implications of these results with respect to the models of parvovirus DNA replication are discussed.     

Evidence for palindromic sequences near the termini of adenovirus 2 DNA

When the kinetics of $\text{Escherichia coli}$ exonuclease III digestion of adenovirus 2 DNA were studied by DNA polymerase I-catalyzed repair synthesis at 5°C, there was an indication of the formation of hairpin structure in the single-stranded template, exposed by exonuclease III. The hairpin structure results from a sequence with an inverted repetition of the type, a b c d···d′ c′ b′ a′. The location of these sequences was determined to be about 180 nucleotides from each terminus of adenovirus 2 DNA with the use of specific restriction endonucleases. The possible role of this region in the replication of the adenovirus 2 genome is discussed.     

The nucleotide sequence recognized by the Escherichia coli K12 restriction and modification enzymes.

The sites recognized by the Escherichia coli K12 restriction endonuclease were localized to defined regions on the genomes of phage φXsK1, φXsK2, and G4 by the marker rescue technique. Methyl groups placed on the genome of plasmid pBR322 by the E. coli K12 modification methylase were mapped in HinfI fragments 1 and 3, and HaeIII fragments 1 and 3. A homology of seven nucleotides in the configuration: 5′-A-A-C .. 6N .. G-T-G-C-3′, where 6N represents six unspecified nucleotides, was found among the DNA sequences containing the five EcoK sites of φXsK1, φXsK2, G4, and pBR322. Three lines of evidence indicate that this sequence constitutes the recognition site of the E. coli K12 restriction enzyme. The C in 5′-A-A-C and the T in 5′-G-T-G-C are locations of mutations leading to loss or gain of the site and thus are positions recognized by the enzyme. This sequence does not occur on φXam3cs70, simian virus 40 (SV40), and fd DNAs which do not possess EcoK sites, and occurs only once on φXsK1, φXsK2, and G4 DNAs, and twice on pBR322 DNA. In order to prove that all seven conserved nucleotides are essential for the recognition by the E. coli K12 restriction enzyme, the nucleotide sequences of φX174, G4, SV40, fd, and pBR322 were searched for sequences differing from the sequence 5′-A-A-C .. 6N .. G-TG-C-3′ at only one of the specified positions. It was found that sequences differing at each of the specified positions occur on DNA sequences that do not contain the EcoK sites. Thus, the recognition site of the E. coli K12 restriction enzyme has the same basic structure as that of the EcoB site (Lautenberger et al., 1978). In each case there are two domains, one containing three and the other four specific nucleotides, separated by a sequence of unspecified bases. However, the unspecified sequence in the EcoK site must be precisely six bases instead of the eight found in the EcoB site. Alignment of the EcoK and EcoB sites suggests that four of the seven specified nucleotides are conserved between the sequences recognized by these two allelic restriction and modification systems.     

Characterization of the ribosome-protected regions of 125I-labelled rabbit globin messenger RNA

The fragments of ¹²⁵I-labelled rabbit globin messenger RNA protected from pancreatic RNAase by initiating 40 S subunits and 80 S ribosomes were analysed using the techniques of RNA sequencing. The fragments were cleaved specifically at cytidine residues generating oligonucleotides labelled in their 3′ terminal residue. Analysis of the partial digestion products of these oligonucleotides after treatment with pancreatic, T₁, U₂ and T₂ RNAase enabled their sequences to be deduced. Sequences were determined from knowledge of the specificities of the ribonucleases and then confirmed in a separate analysis making use of the known electrophoretic mobilities of each base. This combination of methods served to establish that the 40 S- and 80 S-protected fragments are related, and that both contain the initiation codon of the mRNA. The 80 S-protected fragment is about 40 bases in length whilst the 40 S-protected fragments range from 50 to more than 60 bases in length. The most prominent of these 40 S-protected fragments is about 50 bases in length and extends more towards the 5′ end of the mRNA than does the 80 S-protected fragment. It follows that 80 S ribosomes do not protect the 5′ end of the mRNA from nuclease digestion and that the 5′ terminus of rabbit globin mRNA must be at least 15 to 30 bases from the initiation codon.     

Analysis of the cis-acting DNA elements required for piggyBac transposable element excision

The terminal DNA sequence requirements for piggyBac transposable element excision were explored using a plasmid-based assay in transfected, cultured insect cells. A donor plasmid containing duplicate 3′piggyBac terminal inverted repeats was constructed that allowed individual nucleotides or groups of nucleotides within one of the 3′ repeats to be mutated. The relative extent of excision using the mutated end versus the wild-type end was then assayed. Removal of even one of the terminal 3′ G nucleotides from the piggyBac inverted repeat, or removal of the dinucleotide AA from the flanking TTAA target site prevents excision of piggyBac at the mutated terminus. Incorporation of an asymmetric TTAC target site at the 3′ end does not prevent excision from the mutated end. Thus, both piggyBac DNA and flanking host DNA appear to play crucial roles in the excision process.     

A specific endonuclease from Arthrobacter luteus.

A new restriction-like endonuclease, AluI, has been partially purified from Arthrobacter luteus. This enzyme cleaves bacteriophage λ DNA, adenovirus-2 DNA and simian virus 40 DNA at many sites including all sites cleaved by the endonuclease HindIII from Haemophilus influenzae serotype d. Radioactive oligonucleotides in pancreatic DNAase digests of (5′-³²P)-labelled fragments of phage λ DNA released by the action of AluI had the 5′ terminal sequence pC-T-N-. The enzyme recognises the tetranucleotide sequence

and cleaves it at the position marked by the arrows.     

Human immunodeficiency virus integrase protein requires a subterminal position of its viral DNA recognition sequence for efficient cleavage.

Retroviral integration requires cis-acting sequences at the termini of linear double-stranded viral DNA and a product of the retroviral pol gene, the integrase protein (IN). IN is required and sufficient for generation of recessed 3' termini of the viral DNA (the first step in proviral integration) and for integration of the recessed DNA species in vitro. Human immunodeficiency virus type 1 (HIV-1) IN, expressed in Escherichia coli, was purified to near homogeneity. The substrate sequence requirements for specific cleavage and integration of retroviral DNA were studied in a physical assay, using purified IN and short duplex oligonucleotides that correspond to the termini of HIV DNA. A few point mutations around the IN cleavage site substantially reduced cleavage; most other mutations did not have a drastic effect, suggesting that the sequence requirements are limited. The terminal 15 bp of the retroviral DNA were demonstrated to be sufficient for recognition by IN. Efficient specific cutting of the retroviral DNA by IN required that the cleavage site, the phosphodiester bond at the 3' side of a conserved CA-3' dinucleotide, be located two nucleotides away from the end of the viral DNA; however, low-efficiency cutting was observed when the cleavage site was located one, three, four, or five nucleotides away from the terminus of the double-stranded viral DNA. Increased cleavage by IN was detected when the nucleotides 3' of the CA-3' dinucleotide were present as single-stranded DNA. IN was found to have a strong preference for promoting integration into double-stranded rather than single-stranded DNA.     

Specificity determinants for bacteriophage lambda DNA replication: I. A chain of interactions that controls the initiation of replication

The genetic elements which control autonomous DNA replication differ in functional specificity among coliphage λ, the coliphages φ80 and 82, and the Salmonella phage P22. Hybrid phages derived by genetic recombination between λ and each of these related phages have been used to define and to localize specificity determinants for DNA replication.In λ-P22 hybrid phages (Hilliker & Botstein, 1976) the replication control elements segregate as an intact unit. By contrast, some viable λ-φ80 and λ.82 hybrid phages arise by recombination within the replication control region, in a small interval inside structural gene O. From the properties of such hybrid phages, we infer that the O gene product of λ and the functionally equivalent proteins of φ80 and 82 each interact with a specific nucleotide sequence in the cognate ori site, the DNA target for control of the origin of replication. With respect to this interaction, both the O products and the receptor sequences within ori show stringent type specificity. The donor and receptor specificity determinants for the ori-O interaction lie within an interval of less than 400 base-pairs.The O gene product also interacts with the product of replication gene P (Tomizawa, 1971). The O-P interaction displays limited type specificity; the P-like protein of φ80 can function together with the O protein of λ, but the P protein of λ cannot function with the O-like protein of φ80. The specificity determinants for the O-P interaction can be separated from those for the ori-O interaction.We propose that a chain of interactions between ori, O product, P product, and replication functions of the bacterial host, Escherichia coli, controls specific template selection and the assembly of the essential replication apparatus in the initiation of λ DNA replication.     

Patch homologies and the integration of adenovirus DNA in mammalian cells.

The hamster cell line HE5 has been derived from primary hamster embryo cells by transformation with human adenovirus type 2 (Ad2). Each cell contains 2-3 copies of Ad2 DNA inserted into host DNA at apparently identical sites. The site of the junction between the right terminus of Ad2 DNA and hamster cell DNA was cloned and sequenced. The eight [corrected] right terminal nucleotides of Ad2 DNA were deleted. The unoccupied cellular DNA sequence in cell line HE5 , corresponding to the site of the junction between Ad2 and hamster cell DNA, was also cloned; 120-130 nucleotides in the cellular DNA were found to be identical to the cellular DNA sequence in the cloned junction DNA fragment, up to the site of the junction. The unoccupied and the occupied cellular DNAs and the adjacent viral DNA exhibited a few short nucleotide homologies. Patch homologies ranging in length from dodeca - to octanucleotides were detected by computer analyses at locations more remote from the junction site. When the right terminal nucleotide sequence of Ad2 DNA was matched to randomly selected sequences of 401 nucleotides from vertebrate or prokaryotic DNA, similar homologies were observed. It is likely that foreign (viral) DNA can be inserted via short sequence homologies at many different sites of cellular DNA.     

Sequence analysis of the nicks and termini of bacteriophage T5 DNA.

Bacteriophage T5 DNA, when isolated from mature phage particles, contains several nicks in one of the two strands. The 5'-terminal nucleotides at the nicks were labeled with polynucleotide kinase and [gamma-32P]ATP, and the 3'-terminal nucleotides were labeled with Escherichia coli DNA polymerase I and [alpha-32P]dGTP. The sequences around the nicks were analyzed by partial nuclease digestion followed by homochromatography fractionation of the resulting oligonucleotides. The nicks had at least the sequence -PuOH pGpCpGpC- in common. In addition, the two 5' external termini had the first seven nucleotides in common.     

Evidence for two nucleotide sequence orientations within the terminal repetition of adeno-associated virus DNA.

Duplex adeno-associated virus (AAV) DNA, produced by annealing plus and minus virion single strands, has been digested with several bacterial restriction endonucleases. These studies reveal the existence of alternate secondary structures at the termini of duplex AAV DNA. Analysis of the sites of endo R-Hpa II cleavage, the products of complete endo R-Hpa II digestion, and the multiple terminal secondary structures leads to the conclusion that there are two possible nucleotide sequences at each end of AAV DNA. A model that attributes the terminal nucleotide sequence heterogeneity to two possible orientations of the first 120 nucleotides at each end of the DNA is proposed; in one case the sequence is 1 to 120; in the other case the sequence is inverted. An origin of the inversion is suggested based on previously described intermediates in AAV DNA replication.     

Structure of the genome of Moloney murine leukemia virus: a terminally redundant sequence

The genome of the Moloney strain of murine leukemia virus (Mo-MuLV) has been analyzed by digestion with ribonuclease T1 and separation of the digestion products by two-dimensional gel electrophoresis. Thirty large oligonucleotides isolated from such a fingerprint have been characterized. One of these oligonucleotides (number 21) was found to be present in twice the molar yield of the rest. The 30 oligonucleotides were mapped on the genome by determining their yields in various size classes of 3' terminal fragments of Mo-MuLV RNA. The physical map obtained in this way suggested that oligonucletoide 21 was present very near the 3' end of the geome as well as in another location near or at the 5' end. The genome structure suggested by these results was confirmed by analyzing oligonucleotides in Mo-Mulv RNA complementary to strong stop DNA, which is shown to be a copy of the 5' terminal 134 nucleotides of the MoMuLV genome. Some of the oligonucleotides in the RNA protected from RNAase digestion by hybridization to this DNA, including oligonucleotide 21, were present near both the 3' and 5' ends. Comparison of these with the nucleotide sequence of strong stop DNA shows that there is a terminal redundancy of 49-60 nucleotides in the Mo-MuLV genome RNA.