A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.
Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.
A rapid, direct method for screening single plaques of Agt recombinant phage is described. The method allows at least 10(6) clones to be screened per day and simplifies physical containment of recombinants.
With a stepwise degradation and terminal labeling procedure the 3'-terminal sequence of E. coli 16S ribosomal RNA is shown to be Pyd-A-C-C-U-C-C-U-U-A(OH). It is suggested that this region of the RNA is able to interact with mRNA and that the 3'-terminal U-U-A(OH) is involved in the termination of protein synthesis through base-pairing with terminator codons. The sequence A-C-C-U-C-C could recognize a conserved sequence found in the ribosome binding sites of various coliphage mRNAs; it may thus be involved in the formation of the mRNA.30S subunit complex.
pRK212.2, a derivative of the broad host range plasmid RK2, contains two EcoRI cleavage fragments, A and B, neither of which can replicate by itself in Escherichia coli. Fragment A (41.7 kilobases), but not fragment B (14.4 kilobases), can be cloned by insertion into the unrelated plasmids mini-F and ColE1. Fragment B contains the origin of replication and the ampicillin-resistance determinant of RK2. Transformation of E. coli cells containing the mini-F-fragment A hybrid plasmid with fragment B DNA results in the recircularization and replication of fragment B as a nonmobilizable plasmid (pRK2067) with the copy number and incompatibility properties of RK2. Fragment B cannot be cloned in the absence of fragment A because the latter fragment suppresses a function, specified by fragment B, that results in loss of host cell viability. A small segment (2.4 kilobases) of fragment B that contains the RK2 origin of replication but no longer affects host cell growth in the absence of fragment A had been cloned previously by insertion into a ColE1 plasmid. This hybrid plasmid, designated pRK256, will replicate in E. coli polA mutants only when a fragment A-bearing helper plasmid is present. These results demonstrate that the potentially lethal function specified by fragment B of RK2 is not necessary for replication and that at least one trans-acting function is directly involved in RK2 replication.
The strategy of shotgun cloning with M13 is based on obtaining random fragments used for the rapid accumulation of sequence data. A strategy, however, is sometimes needed for obtaining subcloned sequences preferentially out of a mixture of fragments. Shotgun sequencing experiments have shown that not all DNA fragments are obtained with the same frequency and that the redundant information increases during the last third of a sequencing project. In addition, experiments have shown that particular fragments are obtained more frequently in one orientation, allowing the use of only one of the two DNA strands as a template for M13 shotgun sequencing. Two new M13 vectors, M13mp8 and M13mp9, have been constructed that permit the cloning of the same restriction fragment in both possible orientations. Consequently, each of the two strands becomes a (+) strand in a pair of vectors. The fragments to be cloned are cleaved with two restriction enzymes to produce a fragment with two different ends. The insertion of such a fragment into the vector can occur only in one orientation. Since M13mp8 and M13mp9 have their array of cloning sites in an antiparallel order, either orientation for inserting a double-digest fragment can be selected by the choice of the vector.
The restriction endonuclease cleavage sites for SphI and KpnI have been added to the lac cloning region of the phage vectors M13mp10 and M13mp11, using oligodeoxynucleotide-directed in vitro mutagenesis. Complementary deoxy 16-, 21- or 18-mers with the desired base changes were annealed to the M13mp DNA strand and extended with the Klenow fragment of DNA polymerase I. In adding these sites we have shown that this technique can be used as a general method for inserting sequences of DNA as well as introducing deletions and base pair changes.
The sequence of 72 base pairs of the rightward operator (O-R) of bacteriophage lambda is presented as determined with simple and rapid methods for direct DNA sequencing. The sequence of an operator mutant is also described. The methods are of general use in sequencing DNA fragments with unique 5' ends up to 50 base pairs in length. Previous experiments have shown that this operator contains multiple sites recognized by the lambda phage repressor. We believe we have identified three of these sites.
We present a procedure for eucaryotic structural gene isolation which involves the construction and screening of cloned libraries of genomic DNA. Large random DNA fragments are joined to phage lambda vectors by using synthetic DNA linkers. The recombinant molecules are packaged into viable phage particles in vitro and amplified to establish a permanent library. We isolated structural genes together with their associated sequences from three libraries constructed from Drosophila, silkmoth and rabbit genomic DNA. In particular, we obtained a large number of phage recombinants bearing the chorion gene sequence from the silkmoth library and several independent clones of beta-globin genes from the rabbit library. Restriction mapping and hybridization studies reveal the presence of closely linked beta-globin genes.