Interaction of a nuclear protein with a palindromic sequence of the mouse immunoglobulin lambda 2-chain gene promoter is important for its transcription.

By using a gel mobility retardation assay, we detected the formation of three major complexes from the binding of nuclear proteins to the promoter of the immunoglobulin lambda 2-chain gene. Two of the complexes were generated by the presence of an unidentified nuclear factor(s) called herein NF-lambda 2. Although the sequences between lambda 2- and lambda 1-chain gene promoters are very similar, the lambda 1-chain promoter did not compete for the binding of NF-lambda 2 efficiently. The binding site of NF-lambda 2 was localized by DNase I footprinting to a 14-bp region which is about 30 bp upstream of the immunoglobulin octamer motif. This region, referred to as the NF-lambda 2 motif, is within an 18-bp region of twofold rotational symmetry. Experiments with oligomers containing either the NF-lambda 2 or the octamer motifs as competitors for binding and DNase I footprinting, showed that the third complex is the product of the simultaneous binding of an octamer-binding protein and NF-lambda 2. Changing the sequence of the NF-lambda 2 motif to that of the lambda 1-chain counterpart abolished the binding ability of NF-lambda 2. Concomitantly, the level of chloramphenicol acetyltransferase expression driven by the mutated lambda 2 promoter decreased by two- to fivefold when compared with that of the wild-type promoter. It is therefore concluded that the interaction of NF-lambda 2 with the NF-lambda 2 motif stimulates transcription of the lambda 2-chain gene.

Amplification of the gene for histidyl-tRNA synthetase in histidinol-resistant Chinese hamster ovary cells.

Histidinol-resistant (HisOHR) mutants with up to a 30-fold increase in histidyl-tRNA synthetase activity have been isolated by stepwise adaptation of wild-type Chinese hamster ovary (CHO) cells to increasing amounts of histidinol in the medium. Immunoprecipitation of [35S]methionine-labeled cell lysates with antibodies to histidyl-tRNA synthetase showed increased synthesis of the enzyme in histidinol-resistant cells. The histidinol-resistant cell lines had an increase in translatable polyadenylated mRNA for histidyl-tRNA synthetase. A cDNA for CHO histidyl-tRNA synthetase has been cloned, using these histidyl-tRNA synthetase-overproducing mutants as the source of mRNA. Southern blot analysis of wild-type and histidinol-resistant cells with this cDNA showed that the histidyl-tRNA synthetase DNA bands were amplified in the resistant cells. These HisOHR cells owed their resistance to histidinol to amplification of the gene for histidyl-tRNA synthetase.

Structural transitions during maturation of bacteriophage lambda capsids.

The three-dimensional structures of the procapsid and of the mature capsid of bacteriophage lambda were determined to a resolution of approximately 3.4 nm by cryo-electron microscopy and image processing. The mature lambda capsid contains two major proteins, gpE and gpD, arranged on a T = 7 lattice, with gpE arranged as hexamers and pentamers and gpD arranged as trimers. The hexamers and pentamers in the virion display a cartwheel-like structure, with skewed spokes (or arms) radiating out from a central hexameric hub. The thimble-shaped gpD trimers are superimposed on the trivalent interaction point of these arms. A reconstruction of a lambda D- mutant capsid to lower resolution shows no trace of these trimers, thus revealing the interactions of the underlying arms. The procapsid has elongated, irregularly shaped hexamers with gpE subunits set perpendicularly to the capsid surface.

Bacteriophage lambda DNA packaging. The product of the FI gene promotes the incorporation of the prohead to the DNA-terminase complex.

Lambda DNA packaging in vitro can be examined in stages. In a first step, lambda DNA interacts with terminase to form a DNA-enzyme complex, called complex I. Upon addition of proheads, in a second step, a ternary complex, complex II, containing DNA, terminase and the prohead is formed. Finally, upon addition of the rest of the morphogenetic components, complete phages are assembled. We have investigated the effect of the FI gene product (gpFI) in these reactions and found that a stimulation in phage yield is observed when gpFI is included early in the reaction, at the time when DNA, terminase and proheads interact to form complex II. Measurements of complex II formation revealed that gpFI stimulated the rate of formation of this intermediate. gpFI was further shown to stimulate the addition of proheads to preformed complexes I to give complex II, but the protein did not stimulate complex I formation.

Bacteriophage lambda preconnectors. Purification and structure.

The morphogenesis of bacteriophage lambda proheads is under the control of the four phage genes B, C, Nu3 and E, and the two Escherichia coli genes groEL and groES . It has been shown previously that extracts prepared from cells infected with a lambda C-E- mutant accumulate a gpB polymer, which behaves as a biologically active intermediate in prohead assembly. This gpB activity has been called a preconnector , as it is probably a precursor to the head-tail connector. We now report the partial purification of biologically active preconnectors and the characterization of its structure. In the electron microscope, preconnectors appear as donut -like structures composed of several subunits displaying radial symmetry. Optical filtration of periodic arrays of preconnectors showed that the structure has 12-fold rotational symmetry. Side views of the preconnector reveal that it resembles an asymmetrical dumbell . This information has been used to construct a three-dimensional model of the preconnector . The implications of this structure for prohead shape and function, and for DNA packaging are discussed.

Synthesis of a trans-acting inhibitor of DNA maturation by prohead mutants of phage lambda.

Bacteriophage lambda with mutations in genes that control prohead assembly and other head precursors cannot mature their DNA. In this paper we present evidence that the failure of these phage mutants to mature DNA is a reflection of a mechanism that modulates terminase nicking activity during normal phage development. We have constructed plasmids that contain the lambda-cohesive end site (cos) and the genes that code for DNA terminase, the enzyme that matures DNA by cutting at cos. The DNA terminase genes are under control of a thermosensitive cI repressor. These plasmids lack most of the genes involved in prohead morphogenesis and other head precursors. However, when repression is lifted by destruction of the thermosensitive repressor, the terminase synthesized is able to cut almost 100% of the plasmids. Therefore, these plasmids can mature in the absence of proheads and other head gene products. The plasmids are also able to complement mutants of lambda deficient in terminase and DNA maturation. However, in these complementation experiments, if the phage carry mutations in prohead genes E or B, not only is phage DNA maturation blocked, but the plasmid also fails to mature. These experiments show that, in the absence of proheads, phage lambda produces a trans-acting inhibitor of maturation. The genetic determinant of this inhibitor maps in a region extending from the middle of gene B to the end of gene C. A model is proposed in which the nicking activity of DNA-bound terminase is inhibited by the trans-acting inhibitor. Prohead (and other factors) binding to this complex would release the block to allow DNA cleavage and packaging.

Analysis of the expression of murine lambda genes transfected into immunocompetent cell lines.

Hybridoma cell lines were transfected with plasmids containing either a rearranged lambda 1 or a rearranged lambda 2 mouse gene. The levels of lambda-chains synthesized by these transfectants were very low or undetectable. Activation of the expression of the lambda 2 gene was achieved artificially by deleting a portion of the region upstream of the promoter. Analogous deletions in the fragment containing the lambda 1 gene did not result in gene activation suggesting that the upstream sequences of lambda 1 and lambda 2 genes have diverged enough to allow differential regulation of their expression. However, both genes were activated by insertion, at a position upstream of the promoter, of a fragment containing the K-chain gene enhancer. These results suggest that the complete set of sequence elements that mediate lambda gene activation during normal B-cell differentiation are not all contained in the fragments of genomic DNA cloned so far, and thus, at least some of them must be located at a considerable distance from the promoter.

The overproduction of DNA terminase of coliphage lambda.

An artificial operon containing the genes coding for the two subunits of lambda DNA terminase, Nul and A, has been constructed. Derivatives of plasmid pBR322 served as the cloning vehicles. The transcription is driven by the pL promoter of phage lambda, and translation of the terminase genes was made efficient by the replacement of the wild-type ribosome-binding sites for those of lambda genes cII and/or D. The operon also carries the oL operator, and this enables regulation of its expression by a thermosensitive repressor. The synthesis of genes Nul and A products is extremely efficient upon derepression. Within 40 min after induction of the operon, the two subunits comprise about 20% of the total cellular protein mass. Crude extracts prepared from these overproducing strains are at least 100 times more active than extracts prepared from induced lambda lysogens in both promotion of lambda DNA packaging and cosmid cleaving. The ability to produce highly concentrated terminase would enormously facilitate the study of its structure and mechanism of action. These extracts are also extremely useful in techniques such as lambda DNA packaging, cosmid mapping and cosmid linearization to improve efficiency of integration into mouse eggs.

The maturation of coliphage lambda DNA in the absence of its packaging.

In vivo, lambda DNA cannot be cleaved at cos (matured) if proheads are not present; in vitro, however, cos cleavage readily takes place in the absence of proheads. In order to investigate this paradox, we have constructed plasmids that synthesize lambda terminase in vivo upon induction. The plasmids also contain cos at the normal position, about 190 bp upstream of lambda gene Nul. One of the plasmids, pFM3, produces levels of terminase comparable to those found after phage induction. If cells carrying pFM3 are thermoinduced, almost 100% of the intracellular plasmid DNA has a double-strand interruption at or near cos. Since the only lambda genes that pFM3 carries are Nul, A, W and B, this in vivo cleavage is occurring in the absence of proheads. Previous failure to observe lambda maturation with phages carrying prohead mutations may be due to exonucleolytic degradation of the unprotected DNA ends, a different DNA topology or compartmentalization, or terminase inhibition in the absence of prohead by the product of another lambda gene that maps to the right of gene B.

Expression of an immunoglobulin kappa light-chain gene in lymphoid cells using a bovine papillomavirus-1 (BPV-1) vector.

Bovine papillomavirus-1 (BPV-1) replicates extrachromosomally in certain murine cell lines, suggesting that vectors based on the BPV-1 replicon might provide a means of obtaining more uniform gene expression among independent transformants. We have tested such a vector for the expression in hybridoma cells of the immunoglobulin kappa light-chain gene, but found that the level of expression varies greatly among transformants. Our results also indicate that in these transformants the vector has probably been incorporated into chromosomal DNA.

Molecular cloning of an immunoglobulin kappa constant gene from NZB mouse.

An EcoRI fragment carrying the immunoglobulin C kappa gene and multiple J gene segments from the DNA of the NZB strain of mouse was cloned into lambda Ch 4A DNA. Subsequent characterization of the clone by heteroduplex analysis, restriction-enzyme mapping and DNA sequencing demonstrated that the organization of the J gene segments and the C kappa gene of NZB mouse was similar, if not identical, to that of DNA from the Balb/c strain of mouse. Since the amino acid sequence of the light chain of a plasmacytoma of NZB mouse shows a J region sequence different from that of Balb/c mouse, our results indicate that the new J sequence arose by somatic mutation.

Analysis of cosmids using linearization by phage lambda terminase.

A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage lambda terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage lambda (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.

Lethal effect of lambda DNA terminase in recombination deficient Escherichia coli.

lambda DNA terminase is the enzyme that catalyses the cleavage of lambda DNA concatemers into genome-size molecules and packages them into the capsid. The cleavage (DNA maturation) takes place in a specific site in the phage DNA called cos. Either one of two Escherichia coli proteins, integration host factor (IHF) and terminase host factor (THF), is required, in addition to terminase, for maturation of wild-type lambda DNA in vitro. In vivo, at least some cos cleavage is known to occur in mutants that are unable to synthesize active IHF. No THF-defective mutants have yet been isolated. In order to determine if IHF, THF or any other host protein is involved in lambda DNA maturation in vivo, I devised a selection for host mutants that are unable to support cos cleavage. The selection is based on the assumption that lambda DNA terminase will kill cells by cleaving chromosomally located cos sites. I found that DNA terminase will indeed kill cells provided that they contain a chromosomal cos site and provided also that they are defective in the host recA or recB genes. These two genes are required for certain pathways of genetic recombination and repair of damaged DNA, and I suggest that they prevent terminase-induced killing by repairing broken chromosomes. Interestingly, mutation in a related host gene, recD, did not render cells susceptible to terminase killing. recD and recB both encode subunits of exonuclease V, but recD mutants, unlike recB, remain proficient in genetic recombination and repair.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations of the coat protein gene of bacteriophage lambda that overcome the necessity for the Fl gene; the EFi domain.

The functions of most of the 10 genes involved in phage lambda capsid morphogenesis are well understood. The function of the FI gene is one of the exceptions. Mutants in FI fail to mature and package DNA. The gene product (gpFI) seems to act as a catalyst for the formation of an intermediate in capsid assembly called complex II, which contains a procapsid (an empty capsid precursor), terminase (the enzyme that cleaves the DNA precursor and packages it into the procapsid) and DNA. The mechanism for this stimulation remains unknown. It has also been reported that gpFI appeared to stimulate terminase-mediated cos cleavage, in the absence of procapsids, by increasing enzyme turnover. In comparison with other head-gene mutants, FI mutants are leaky, producing approx. 0.1 phage per infected cell. Some second-site revertants of FI- phages, called 'fin', that bypass the necessity for gpFI, have been isolated and found to harbour a mutation in the genes that code for the two subunits of terminase. In the course of mapping additional fin mutants, it was discovered that some mapped outside the terminase genes. To localize the mutations, restriction fragments of fin mutant DNAs were subcloned into plasmids and their ability to contribute to fin function was determined by marker-rescue analysis. The location of the fin mutation was further delineated by deletion analysis of a plasmid that was positive for fin. This showed that some fin mutations mapped to a region comprising genes E, D and a portion of C. The sequencing of this entire region in several fin isolates showed that the fin mutations are clustered in a small region of gene E corresponding to a portion of 26 amino acid residues of the coat protein (gpE). We have called this region of the protein the EFI domain. All the mutations result in an increase in positive charge relative to the wild-type protein. These results suggest that DNA maturation and packaging are in part controlled by an interaction between gpFI and capsid gpE.

Early intermediates in bacteriophage lambda prohead assembly. II. Identification of biologically active intermediates.

The morphogenesis of bacteriophage lambda proheads is under the control of the four phage genes B, C, Nu3, and E, as well as the E. coli genes groEL and groES. It has been previously shown that extracts prepared from cells infected with a lambda C-E- mutant accumulate biologically active gpB and gpNu3 (Murialdo, H., and Becker, A., J. Mol. Biol. 125, 57-74 (1978) ). To characterize the nature of these intermediates in prohead assembly, extracts prepared from these cells were fractionated by DEAE-cellulose chromatography as well as velocity sedimentation. Intermediates containing gpB were identified by SDS-polyacrylamide gel electrophoresis and by their ability to be assembled into biologically active proheads in vitro. The results indicate that the most abundant, biologically active intermediate (greater than 98% of the gpB activity) is a 25 S gpB-containing polymer. A second biologically active intermediate (about 1% of the total gpB activity) was identified as a gpB-gpgroEL complex.

Stimulation of groE synthesis in Escherichia coli by bacteriophage lambda infection.

We found that infection of Escherichia cell by lambda results in at least a twofold stimulation in the rate of synthesis of one of the products of groE. To determine what lambda-coded factors were responsible for this stimulation, numerous phage lambda mutants carrying bio substitutions were analyzed for their ability to stimulate groE synthesis. Our results revealed that the main factor(s) which is responsible for stimulating groE synthesis is located between the endpoints of the lambda bio69 and lambda bio252 substitutions, a region of DNA coding for bet, gam, kil, and cIII.

Assembly of biologically active proheads of bacteriophage lambda in vitro.

Bacteriophage lambda DNA can be packaged in vitro into preformed proheads to generate plaque-forming units. This complex set of reactions is initiated when lambda DNA is mixed with the product of the phage A gene, and proheads. Because proheads are an essential early reactant, the system has potential as an assay for the formation of biologically active proheads. When extracts of cells infected with certain lambda head mutants (for example, B--, C--, Nu3--, and E--) are used as the prohead donor, plaque-forming units are not produced. However, when extracts of E- - and Nu3- - infected cells are first reacted together the combination provides prohead-donor activity to the in vitro packaging system. In vitro assembled, biologically active proheads have the same sedimentation properties and electron micrsocopic appearance as "wild-type" proheads isolated from lambdaA-D- -infected cells. Centrifugation analysis shows that the Nu3- extract contributes gpE, the major capsid protein, to the reaction in the form of monomers or small polymers.