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New phage lambda-based cloning vectors, Charons 32, 33, 34 and 35, have been constructed. These vectors allow cloning of large (19-21 kb) DNA fragments in up to six cloning sites. DNA cloned in these vectors can be propagated on recA- Escherichia coli hosts.

DNA replication in bacteriophage lambda begins at a unique origin between residues 39,000 and 39,200 of the lambda genome. This segment of DNA serves a dual function since it also lies within the coding sequence of the lambda replication initiator protein O which binds origin DNA. The lambda origin sequence contains four 19-base-pair (bp) segments (iterons) which have dyad symmetry, followed by a 40-bp A + T-rich zone of highly asymmetrical base composition. It was noted earlier that lambda origin DNA exhibits an anomalous electrophoretic mobility on gels; that is, the length of DNA as determined by DNA sequencing is approximately 20% less than is predicted from electrophoretic mobility. Recent studies of kinetoplast minicircle DNA (K-DNA) from the protozoan Leishmania tarentolae have led to the proposal that sequence-induced DNA curvature could account for such electrophoretic anomalies by alteration of the shape of the DNA molecule. We now present evidence that the lambda origin contains a static curve.

A transducing phage has been isolated with codes for the sigma subunit of Escherichia coli RNA polymerase. Transducing phage were selected from E. coli shotgun collections of HindIII or Sac I fragments cloned into Charon 25, a new bacteriophage lambda vector that is capble of forming lyosogens at high temperature. Transduction of an E. coli strain carrying a temperature-sensitive mutation in the sigma gene was used for the selection. The positions of restriction sites for Sac I, HindIII, Xho I, Bgl II, and Kpn I in the cloned bacterial DNA segments were determined. Phage containing the HindIII fragment complement both primase (dnaG) and sigma (rpoD) whereas those containing the Sac I fragment complement only sigma. Results of analyses of the proteins made both in vivo after infection of UV-irradiated cells and in vitro in a coupled transcription/translation system suggest that a Sac I site separates the promoter for sigma from the sigma structural gene. The direction of transcription of sigma was determined to be clockwise with respect to the E. coli genetic map.

A plasmid has been constructed, pOri2, which contains two lambda replication origin sequences separated by 1068 bp; both lambda sequences having the same orientation. When lambda initiation protein O is reacted with linearized pOri2 and examined by electron microscopy it is found to contain a looped area in which two parts of the plasmid are bound together by the O protein complex. Length measurements show that the O protein binds at the expected positions of the lambda origin sequences and that the looped area represents the DNA segment between the two O protein binding domains. Similar looping occurs in reactions with supercoiled pOri2 or if an amino-terminal fragment of O protein is used. When looped molecules are reacted with psoralen, crosslinked by irradiation with uv light, and then denatured, it is found that the looped area is more thermostable than the rest of the molecule. This indicates that the DNA within the looped segment is torsionally constrained while that outside the loop is free to rotate and suggests that simultaneous binding of O to two origins fixes the linkage number of the intervening DNA. The double origin binding ability of O may be diagnostic of the details of the reaction of O with a single origin sequence. A model is presented that rests on the assumption that O can produce microscopic looping between O protein binding sites within a single ori sequence.

DNA fragments from lambdaspc1 and lambdafus2, carrying ribosomal protein genes from Escherichia coli, were inserted into lambda phage vectors Charon 3 and Charon 4. Eight of the resulting clones were characterized by agarose gel electrophoresis of EcoRI digests, analytical CsCl equilibrium centrifugation, and electron micrographic analysis of heteroduplexes. In each case, the identity, order, and orientation of each cloned fragment was determined. In all, 8 of the 12 EcoRI fragments of lambdafus2 were cloned in various arrangements. In the accompanying paper, genes for 15 ribosomal and related proteins and three bacterial promoters were detected in these phages. In addition, four of the hybrid phages carried fragments of lambda-DNA including the phage origin of replication (ori), the late promoter, PR', and the cohesive ends (cos site) in both orientations. The latter phages yield a circularly permuted collection of DNA molecules.

A single DNA fragment containing both mu and delta immunoglobulin heavy chain genes has been cloned from normal BALB/c mouse liver DNA with a new lambda phage vector Charon 28. The physical distance between the membrane terminal exon of mu and the first domain of delta is 2466 base pairs, with delta on the 3' side of mu. A single transcript could contain a variable region and both mu and delta constant regions. The dual expression of immunoglobulins M and D on spleen B cells may be due to alternate splicing of this transcript.

The Charon lambda bacteriophages have been developed as vectors for cloning. Their construction incorporates mutations that make them simple to use and also greatly increases their safety for the biological containment of cloned recombinant DNA. Three of the Charon vector phages, 3A, 4A, and 16A, have been certified for use as EK2 vector-host systems, when propagated in bulk in a special bacterial host, DP50SupF. We present here some of the data on which the safety of these systems was evaluated. DNA fragments ranging in size from 0 to 2.2 X 10(4) base pairs can be cloned in these EK2 Charon phages.

Specific DNA restriction endonuclease fragments can be identified after electrophoresis in agarose gels by hybridization in the gel (in situ) to radioactive homologous RNA. RNA-DNA hybrids are detected by autoradiography of the gel. Comparison of band patterns of the autoradiogram and the ethidium bromide stained gel allows the identification of the DNA fragment which is complementary to the RNA probe. The technique is rapid, easy and inexpensive. It is sensitive enough to detect individual genes in a mixture of fragments produced by restriction enzyme digestion of complex cellular DNA. We have used this technique to determine which of the Hin III and Eco R1 fragments of phi80d3ilv+su+7 and E. coli DNAs contain the 5S, 16S and 23S ribosomal RNA (rRNA) genes of E. coli.

We have characterized the binding of lambda phage replication initiation protein O to the phage origin of replication. The minimal DNA segment required for O binding is the single iteron, a 19-bp sequence of hyphenated dyad symmetry that is repeated with variations four times in the origin. The isolated amino terminus of O protein is also sufficient to bind DNA. Electrophoretic studies show that the amino terminus of O protein induces bending of a single iteron. The DNA-protein interaction was characterized by ethylation interference, dimethyl sulfate protection and neocarzinostatin footprinting. Points of DNA-protein contact are largely concentrated in two areas symmetrically disposed with respect to the dyad symmetry of the iteron. This suggests the protein interacts as a dimer with half sites in the DNA. However, a few non-symmetrical contacts are found, indicating that O protein may distort the helix. This may correlate with the bending effects demonstrated electrophoretically. Cylindrical DNA projections were used to model O protein binding to the lambda origin and compare it with the lambda repressor-operator interaction. Whereas bound repressor nearly encircles the DNA in the major groove, O protein leaves the major groove on the opposite side exposed.

The DNA coding for the human immunoglobulin D(IgD) heavy chain (delta, delta) has been sequenced including the membrane and secreted termini. Human delta, like that of the mouse, has a separate exon for the carboxyl terminus of the secreted form. This feature of human and mouse IgD distinguishes it from all other immunoglobulins regardless of species or class. The human gene is different from that of the mouse; it has three, rather than two, constant region domains; and its lengthy hinge is encoded by two exons rather than one. Except for the third constant region, the human and mouse genes are only distantly related.

Experiments have been performed to help clarify the role of nonhomologies in phage lambda recombination. Three-factor crosses were carried out, and the frequencies of single and double recombinants in the two adjoining intervals were compared when the central marker was either a double point mutation (v1v3) or deletion (rex-cI deletion) or nonhomologous substitution (imm434). In all cases the lefthand marker was a bio substitution (Fec- phenotype, which does not permit plating on recA-), and the righthand marker was an amber mutation in gene O. Experiments were performed in all four possible arrangements of the central and rightward markers, while selecting for the Fec+ phenotype on the recA- host. As anticipated, high negative interference (HNI) was observed with point mutations, but when the central marker was a substitution nonhomology, HNI was reduced about tenfold. Surprisingly, when the central marker was a simple deletion, a dramatic asymmetry in results was observed, with HNI being exhibited only when the central deletion marker was acquired by the double recombinant. These results indicate that under normal conditions (red+, gam+, rec+) and with noninhibited DNA replication, recombination in coliphage lambda entails a highly asymmetric step that could be at the level of strand transfer or mismatch repair.

The Ti plasmid sequences (T-DNA) from the octopine-producing crown gall tumor A6S/2 were isolated by molecular cloning, using the bacteriophage lambda vector Charon 4A. Analysis of the cloned DNA segments indicates that the Ti plasmid sequences are covalently joined to plant nuclear DNA. These data demonstrate that genetic recombination between a eukaryote and a prokaryote can occur as a natural phenomenon.

A map of bacteriophage lambda was constructed, including accurate positions for all 41 cut sites made by 12 different restriction enzymes. Over 100 fragments from single, multiple, and partial enzyme digestions were measured versus standards that were calibrated with respect to DNA molecules of known sequence. The data were subjected to least-squares analysis to assign map coordinates. In no case did a fragment size predicted from the map differ from the measurement of the fragment by more than +/- 5%. This low error rate was consistent in all size ranges of fragments. The total length of lambda was calculated as 49,133 nucleotide pairs. This probably is accurate to within 500 base pairs.

The complete nucleotide sequence of the gamma 2b constant region gene cloned from BALB/c liver DNA is reported. The sequence of approximately 1870 base pairs includes the 5' flanking, 3' untranslated, and 3' flanking regions and three introns. The C gamma 2b coding region is divided by these introns into four segments corresponding to the homology domains and hinge region of the protein. The introns separating the hinge from the CH2 domain and the CH2 from the CH3 domain are small (106 and 119 base pairs). A larger intervening sequence of 314 base pairs separates the CH1 and hinge regions. The stretch of DNA comprising this large intron plus the hinge shows a strong homology with the other CH domains.

We describe the isolation of a new class 2 restriction endonuclease from Providencia stuartii 164. Using the procedure of osmotic shock treatment, we have partially purified this enzyme (Pst 1) and have begun preliminary work to characterize its specificity and requirements. Pst 1 requires Mg++ as the only cofactor and produces more than 18 cleavages in wild type lambda. We have determined the location of 7 of these cleavages by the use of deletion and insertion mutants of lambda.

A 15,8-kilobase pair fragment of BALB/c mouse liver DNA, cloned in the Charon 4A lambda phage vector system, was shown to contain the mu heavy chain constant region (CHmu) gene for the mouse immunoglobulin M. In addition, this fragment of DNA contains at least two J genes, used to code for the carboxyl terminal portion of heavy chain variable regions. These genes are located in genomic DNA about eight kilobase pairs to the 5' side of the CHmu gene. The complete nucleotide sequence of a 1120-base pair stretch of DNA that includes the two J genes has been determined.

A computer language termed DNA has been devised to aid in the description of DNA sequence manipulations. This was an outgrowth of a DNA sequence editor which has been implemented for a microcomputer. A formal description of the language in the BNF formalism is presented.

The molecular structure of a mouse immunoglobulin D from a plasmacytoma tumor and that of the normal mouse gene coding for immunoglobulin D are presented. The DNA sequence results indicate an unusual structure for the tumor delta chain in two respects: (i) Only two constant (C) region domains, termed C delta 1 and C delta 3 by homology considerations, are found; the two domains are separated by an unusual hinge region C delta H that lacks cysteine residues and thus cannot provide the covalent cross-links between heavy chains typically seen in immunoglobulins. The two domains and hinge are all coded on separate exons. (ii) At the carboxyl end of the delta chain there is a stretch of 26 amino acids that is coded from an exon located 2750 to 4600 base pairs downstream from the rest of the gene. Analogy with immunoglobulin M suggests that this distally coded segment C delta DC may have a membrane-binding function; however, it is only moderately hydrophobic. A fifth potential exon (C delta AC), located adjacent to the 3' (carboxyl) end of C delta 3, could code for a stretch of 49 amino acids. The tumor's expression of the delta gene may be aberrant, but the simplest interpretation would be that this tumor expresses one of the several biologically significant forms of the delta chain.

A comprehensive package of software has been developed to access nucleic acid and protein sequence databases on stand-alone IBM personal computers. The software combines keyword search on the annotation fields of the data with pattern matching algorithms on the biological sequences. Sequences containing complex sites like promoters or kink sites can be identified as well as sequences that are similar to a query sequence. Protein sequences with particular patterns of amino acids such as hydrophobic regions can be identified as well. Considering the relatively inexpensive hard disks now available, personal computers have become a cost-effective alternative to mainframe processing for sequence databases.

The interaction of the lambda phage initiator protein, O, with the lambda origin sequence, ori, has been investigated. Binding of O, or its amino-terminal fragment, causes a major structural change within a 60 bp AT-rich region just to the right of the O-binding site. ATP or other molecular energy sources are not required. The modification, as assayed by nuclease sensitivity, is reduced when certain ori mutant sequences, which bind O but fail to replicate, are substituted for the wild-type sequence. The modification of DNA structure caused by the interaction of O is absolutely dependent on the presence of superhelical tension at the lambda origin sequence, and has several properties consistent with a strand separation reaction. We propose that this modification is a fundamental prepriming event that is the first stage in initiation of bidirectional replication in lambda after O binding.

Analysis of the region 3' to the CD4+ T-cell gene Rpt-1 (encoding regulatory protein T-lymphocyte 1) led to the definition of a silencer element that inhibits heterologous gene expression in certain CD4+ T-cell lines but not in B-cell or non-lymphoid cell lines. Functional silencer activity in vivo was associated with the presence of a specific silencer-DNA-protein complex in electrophoretic mobility shift assays with T-cell extracts. Formation of this complex was selectively inhibited by the region in HIV-1 containing a silencer element. We discuss the possibility that DNA-binding factors may coregulate HIV-1 and Rpt-1 gene expression through a common transcriptional silencer element.

The prevalent forms of adult and childhood B-cell neoplasia are chronic lymphocytic (CLL) and acute lymphocytic (ALL) leukaemia, and are typified by a nearly monoclonal accumulation of cells expressing a single heavy (H) and light (L) chain variable (V) region. V gene selection could be random, or quite biased if the disease or the developmental status of the transformed cell somehow influenced DNA rearrangement. We have cloned and sequenced three germ-line VH gene segments that constitute a new human VH family (subgroup V) linked within 160 kilobase pairs of the DH-JH complex. One VH(V) member is rearranged in about 30% of patients with CLL and ALL, but not in IgM-expressing B-cell lines from peripheral blood. In some tumours, we detect a truncated (VH(V) RNA devoid of constant regions that originates from unrearranged VH(V) genes. In other tumours and in resting splenocytes, we detect large amounts of normally sized VH(V)-associated mRNA, although stimulation by mitogen of splenic B cells results in loss of VH(V)-hybridizing RNA. These features suggest that biased rearrangement of subgroup V may be under developmental selection.

A fragment of bacteriophage lambda DNA produced by the restriction endonuclease Eco RI and extending from the immunity region to a point inside gene O is found to have a fully functional origin of replication. Seven ori- mutations of lambda cluster in a small region just to the left of the Eco RI cleavage site which defines the right end of this fragment. These mutations lie within gene O.

Whether the immunoglobulin (Ig) heavy chain genes C mu and C delta are expressed singly or in combination, their transcripts undergo differentiation-specific alterations in membrane (M) versus secreted (S) forms as well as in abundance. To better understand this regulation, we have cloned cDNAs for human delta m and delta s to establish the 3' end of the C mu-C delta transcription unit. Steady state mRNA levels and transcription rates were then analyzed in normal and transformed human B cells representing different maturation and activation states. The ratio of micron/microsecond RNA and of delta m/delta s RNA correlated with developmental stage, with a higher ratio at earlier stages. Steady state ratios of total mu/delta RNA paralleled ratios of C mu/C delta nascent transcription, suggesting no major posttranscriptional control for differential expression. However, at all developmental stages, transcription termination occurred downstream of the micron exons, suggesting a strong posttranscriptional regulatory component for production of secreted versus membrane forms of mu RNA. The relative abundance of mature delta S RNA was considerably higher in the human than in the mouse, correlating with the increased levels of circulating IgD in the former species. Stimulation of human splenocytes with mitogens did not increase delta RNA; in fact, splenocytes activated with pokeweed mitogen were nearly devoid of delta RNA, and Staphylococcus aureus Cowan I caused only a minor change.

New phage lambda based cloning vectors, Charons 36-40, have been constructed which allow cloning of large (up to 24 kb) DNA fragments with up to sixteen cloning enzymes. Several of these could not be used previously with lambda vectors. Clones produced with these vectors can be propagated under recombination deficient conditions. A novel polystuffer method has been developed that permits vector arms to be purified by simple precipitation and which allows reliable identification of clones that have reincorporated any part of the stuffer. Three of the vectors are available with amber mutations in essential genes.

A simple method is described for speeding up the computation of base pairing interractions. It is especially effective on microcomputers.

Segments of the replication control region of bacteriophage lambda (lambda) and lambda mutants defective in replication were attached in vitro to the phi80 phage vector Charon 3 and to the plasmid vector mini Col El (pVH51). The chimeric phages and plasmids have been used to localize the origin of lambda DNA replication and to facilitate a structural analysis of the lambda replicator.

The entire nucleotide sequence (approximately 20 kbp) spanning the human immunoglobulin IgM (mu) and IgD (delta) heavy chain constant region genes has been determined from DNA of mu-delta producing chronic lymphocytic leukemic B cells. As in the murine IgM + IgD double-producing B cells, no rearrangement has occurred in the C mu-C delta region in the leukemic cells. The C mu locus is highly conserved between mouse and human with the exception of the nucleotide sequence between the C mu 4 and mu M1 exons, which has diverged dramatically. The intergenic sequence between human C mu and C delta is three times larger than the analogous region in the mouse and contains notable features absent from the mouse, including a 443 bp segment that is 96% identical to a 442 bp sequence that occurs just 3' to the heavy chain enhancer, a 366 bp sequence that is directly repeated with 76% homology, and 12 tandem copies of a 35 bp sequence. The human C delta gene contains two additional exons relative to mouse C delta, but shares with the mouse the unique distal location of both secreted and membrane coding segments. Several polymorphisms in the human population have been identified in the intergenic region and in C delta but not in C mu.

The delta heavy (H) chain of mouse immunoglobulin D (IgD) is unusual both in its structure and in its differential expression relative to immunoglobulin M (IgM; reviewed in ref. 1). The region of DNA between IgM and IgD H-chain constant-region genes is probably implicated in this control. So far only fragments of the area have been sequenced. Now, however, we present the complete sequence as well as the sequence of the introns of the C delta gene. We have found several interesting features (Fig. 1), including an open reading frame (ORF) between Cmu and C delta which encodes 146 amino acids that might represent a previously unsuspected domain-like protein; three blocks of simple repetitive sequences; a 162-base pair (bp) unique-sequence inverted repeat; and a domain-like pseudogene in the large intron of C delta. We have not found, however, any sequence 5' of C delta resembling the switch (S) recombination sequences associated with class switching in other heavy chains. Moreover, we have determined the 3' deletion end point of an IgD-producing myeloma and find no sequences reminiscent of switch sites nearby.

TEPC 1017 is a BALB/c plasmacytoma that synthesizes IgD in large enough amounts to permit the isolation of mRNA for mouse delta chains. cDNA has been prepared from this mRNA, and an 880-base-pair fragment of it has been cloned by recombinant DNA techniques. The hybridization selection technique has been used to show that this cDNA clone specifically binds only mRNA that can be translated into immunoprecipitable delta chains. the sequence of a portion of this clone has been determined and, when translated, shows homology with the C delta 3 of a human myeloma protein. Using this cDNA clone as a probe, we have found that several different-sized delta RNAs are present in TEPC 1017 and in another IgD-secreting plasmacytoma, TEPC 1033.