Molecular dissection of the murine antibody response to streptococcal group A carbohydrate.

Monoclonal antibodies (mAb) to streptococcal group A carbohydrate (GAC) are encoded by a minimum of two VH, four JH, four V kappa, three J kappa, one V lambda, and one J lambda gene segments. The IdX, IdI-1, and Id5 idiotypic determinants are expressed by anti-GAC mAb and are found on free kappa chains. Each pattern of these determinants is encoded by a distinct V kappa gene segment, apparently without the requirement for a particular J kappa, VH, or JH gene segment, or somatic mutation. In contrast, the binding site-associated idiotypic determinant IdI-3a does not correlate with any single V or J gene segment.

Sindbis virus: an efficient, broad host range vector for gene expression in animal cells.

Sindbis virus, an enveloped virus with a single-stranded RNA genome, was engineered to express a bacterial protein, chloramphenicol acetyltransferase (CAT), in cultured insect, avian, and mammalian cells. The vectors were self-replicating and gene expression was efficient and rapid; up to 10(8) CAT polypeptides were produced per infected cell in 16 to 20 hours. CAT expression could be made temperature-sensitive by means of a derivative that incorporated a temperature-sensitive mutation in viral RNA synthesis. Vector genomic RNAs were packaged into infectious particles when Sindbis helper virus was used to supply virion structural proteins. The vector RNAs were stable to at least seven cycles of infection. The expression of CAT increased about 10(3)-fold, despite a 10(15)-fold dilution during the passaging. Sindbis virus vectors should prove useful for expressing large quantities of gene products in a variety of animal cells.

Application of latex microspheres in the isolation of plasma membranes. Affinity density perturbation of erythrocyte membranes.

Immunolatex spheres, originally developed as visual markers for scanning electron microscopy, were employed as membrane density perturbation reagents. Methacrylate spheres were bound to antibody molecules and used to label antigens on erythrocytes. Ghosts prepared from labeled cells were subjected to isopycnic centrifugation on continuous sucrose and dextran gradients. It was found that the labeled erythrocyte membranes had a substantially higher density than unlabeled membranes. The extent to which the membrane density was shifted on a given gradient depended on the number, size and density of the latex spheres and could be closely predicted by theory. These results suggest that the reagents and techniques described here have potential application for the isolation of plasma membranes from more complex cell types.

Homologous recombination in Escherichia coli: dependence on substrate length and homology.

We studied the in vivo recombination between homologous DNA sequences cloned in phage lambda and a pBR322-derived plasmid by assaying for the formation of phage-plasmid cointegrates by a single (or an odd number of) reciprocal exchange. (1) Recombination proceeds by the RecBC pathway in wild-type cells and by low levels of a RecF-dependent pathway in recBC- cells. The RecE pathway appears not to generate phage-plasmid cointegrates. (2) Recombination is linearly dependent on the length of the homologous sequences. In both RecBC and RecF-dependent pathways there is a minimal length, called the minimal efficient processing segment (MEPS), below which recombination becomes inefficient. The length of MEPS is between 23-27 base pairs (bp) and between 44-90 bp for the RecBC- and RecF-dependent pathways, respectively. A model, based on overlapping MEPS, of the correlation of genetic length with physical length is presented. The bases for the different MEPS length of the two pathways are discussed in relationship to the enzymes specific to each pathway. (3) The RecBC and the RecF-dependent pathways are each very sensitive to substrate homology. In wild-type E. coli, reduction of homology from 100% to 90% decreases recombinant frequency over 40-fold. The homology dependence of the RecBC and RecF-dependent pathways are similar. This suggests that a component common to both, probably recA, is responsible for the recognition of homology.

Sindbis virus vectors for expression in animal cells.

Sindbis virus and other alphavirus gene expression vectors have recently been used to express and study the functions of proteins and RNA, to evaluate classical vaccine and novel antiviral approaches, and for nucleic acid immunization. The vectors will likely attract continuing, innovative applications that exploit their useful features: rapid and efficient gene expression, wide host range, and RNA genomes.

Bidirectional chain-termination nucleotide sequencing: transposon Tn5seq1 as a mobile source of primer sites.

The sequencing of large DNA fragments by the chain-termination method [Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467] has generally required extensive manipulations to bring all parts of the fragment near a specific primer-binding site, or the repeated synthesis of new oligodeoxynucleotide primers. Here we develop a more efficient approach, the use of a transposable element to insert primer binding sites at random in the DNA of interest. We constructed a Tn5 derivative called Tn5seq1 with unique DNA segments near each end so that oligodeoxynucleotides matching them could serve as primers for sequencing in each direction from any Tn5seq1 insertion site. Our experiments demonstrate the use of Tn5seq1 for sequencing in pBR322 plasmids and also in uncloned DNAs of the Escherichia coli chromosome. The unique segments near the left and right ends of Tn5seq1 are promoters from phages T7 and SP6, respectively, to permit the efficient transcription of adjacent DNAs in vivo or in vitro.

Utilization of heterologous alphavirus junction sequences as promoters by Sindbis virus.

We used Sindbis virus, an alphavirus, as a model to study the evolution of the recognition of viral cis-acting sequences. During the life cycle of alphaviruses, a full-length minus-strand RNA is made and serves as a template for both genomic RNA replication and subgenomic mRNA transcription. Transcription initiates at an internal promoter site, the junction sequence, to produce a subgenomic mRNA. The junction sequences of alphaviruses are highly conserved, but they do contain a number of base differences. These could have been essentially neutral mutations during evolution, such that any of the contemporary sequences can be recognized efficiently by any of the alphaviruses. Alternately, the changes could have resulted in significant functional divergence, such that the contemporary viruses can no longer recognize heterologous junction sequences as promoters. To distinguish between these possibilities, we constructed Sindbis virus derivatives with two subgenomic mRNA promoters. One is the wild-type Sindbis virus promoter used for expression of the structural proteins. The other is either the minimal Sindbis virus promoter or the corresponding junction sequences from other alphaviruses, which are placed upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene. RNA analyses were used to determine the relative promoter strengths of the various junction sequences. The results showed that all but two were recognized as promoters by Sindbis virus. CAT enzyme assays were used to measure the accumulation of CAT protein made from mRNAs transcribed by using the heterologous junction sequences as promoters. Most of the viruses expressed amounts of CAT enzyme within 10-fold of each other. The two viruses with junction sequences that were not recognized as promoters did not give significant CAT expression. We conclude that, with respect to Sindbis virus, the junction sequences are functionally conserved; i.e., most of the contemporary nucleotide differences in the junction sequences are neutral or near-neutral mutations. The functional conservation suggests that neither the cis-acting sequence nor the cognate binding site of the transcription factor can change independently. This type of coupled evolution between cis-acting sequences and their cognate viral protein binding sites may be a general phenomenon. For example, it explains the ubiquitous presence of conserved cis-acting sequences in each of the families of RNA viruses. There are implications of this hypothesis for the design of antiviral drugs.

Analysis of Sindbis virus promoter recognition in vivo, using novel vectors with two subgenomic mRNA promoters.

Four types of Sindbis virus vectors, each carrying two promoters for subgenomic mRNA synthesis, were designed to measure relative promoter strengths and to survey potential contextual effects on promoter strengths. One of the promoters in each vector was used as the reference promoter, while the other was the one being tested. We used these vectors to measure the relative strengths of four promoters: the minimal promoter, an extended sequence believed to have full promoter activity, and two mutant promoters, one with an inactivating 3-nucleotide insertion called CR4.1 and the other with a 4-nucleotide deletion called delta 4. The strengths of the promoters were measured by quantitating the RNA transcribed from each promoter in vivo and also by assaying for chloramphenicol acetyltransferase activity encoded by one of the two transcripts. We found that the relative strengths of the promoters were similar in different contexts. The complete promoter was 6-fold more active, the delta 4 promoter was (surprisingly) about twice as active, and the CR4.1 promoter was 100-fold less active than the minimal promoter. At least two contextual effects were identified that can alter the activity of one or both promoters in the vectors. One effect is that given identical promoters, the 3'-proximal promoter on the minus-strand template can be more active than the 5'-proximal promoter. This may be due to preferential association of one or more components of the transcription complex for the 3' end of the minus-strand template. A second effect is promoter competition, particularly when the promoters are closely spaced.

Evolution of the Sindbis virus subgenomic mRNA promoter in cultured cells.

Transcription of the subgenomic mRNA of alphaviruses initiates at an internal site, called the promoter, which is highly conserved. To determine the functional significance of this conservation, we used an approach that randomizes positions -13 to -9 of the promoter to generate a library containing all possible sequences within this region, including the wild-type sequence. Viruses in the mixed population with more-efficient promoters were selected for during passaging in mammalian (BHK-21) cells. Results from early passage populations indicate that a large number of different promoters are functionally active. Analysis of eight individual viruses found that although each contained a promoter with different degrees of sequence identity to the wild-type sequence, all eight viruses produced progeny. This suggests that the mechanism for transcription allows for a diversity of sequences to serve as promoters. Further passaging of the viral library led to a population consensus sequence that increasingly resembled the wild-type sequence, despite the fact that these promoters are not constrained by the need to encode the carboxyl terminus of the nsP4 protein. Thus, conservation of the region of the promoter from -13 to -9 is in large part due to selection for promoter function, and the wild-type sequence and sequences closely similar to it seem to be optimal for promoter function in BHK-21 cells.

Host-dependent evolution of the Sindbis virus promoter for subgenomic mRNA synthesis.

Alphaviruses are alternately transmitted between arthropod and vertebrate hosts. In each host, the virus transcribes a subgenomic mRNA that encodes the viral structural proteins which encapsidate the genome to form progeny virions. Transcription initiates at an internal site called the promoter. To determine if promoter utilization varies in mammalian versus mosquito cells, we used these cells as hosts to select for active promoters among a library of different mutant promoters. Compared with that in BHK-21 cells, selection was more rapid in mosquito (C7-10) cells, with much less diversity of promoters remaining after fewer passages. Thus, promoter selection is host dependent. With further passaging, both BHK-21 and C7-10 cells selected for similar sequences that closely resemble the wild-type promoter sequence. The difference in the rates of selection is not because BHK-21-derived promoters cannot function in mosquito cells. Instead, part of the host dependence is probably due to posttranscriptional differences between BHK-21 and C7-10 cells that may require more active promoters in mosquito cells. Part of the host dependence may also be attributed to the decreased rate of transcription versus that of replication in mosquito cells. This change in regulation of subgenomic to genomic RNA synthesis appears to correlate with the extent of cleavage or pausing of the genomic RNA synthesis at or close to the promoter.

A novel viral RNA species in Sindbis virus-infected cells.

Sindbis virus (SIN), the type alphavirus, has been studied extensively to identify the viral cis-acting sequences and proteins involved in RNA transcription and replication. However, very little is known about how these processes are coordinated. For example, synthesis of the genomic RNA and the subgenomic mRNA depends on the minus strand. Do these activities occur independently on different templates, or can replication and transcription take place simultaneously on the same template? We describe the appearance of a SIN-specific, plus-sense RNA that is intermediate in size between the genomic and subgenomic RNA species. This RNA, designated RNA II, is observed in a number of different cell lines, both early and late in infection. The number of RNA II species, their sizes, and their abundances are influenced by the subgenomic promoter. We have mapped the 3' end of RNA II to a site within the subgenomic promoter, four nucleotides before the initiation site of the subgenomic mRNA. Our results indicate that the appearance of RNA II is correlated with subgenomic mRNA transcription, such that strong or active promoters tend to increase the abundance of RNA II, relative to weak or less active promoters. RNA II is most abundantly detected with the full promoter and is at much lower abundance with the minimal promoter. The possible origins of RNA II are discussed.

Effect of base pair mismatches on recombination via the RecBCD pathway.

The effect of base pair mismatches on recombination via the RecBCD pathway was studied in mutS and wild-type Escherichia coli, using substrates that contain single or multiple mismatches. Recombination between homologous DNA inserts in lambda phage and pBR322-derived plasmids forms phage-plasmid cointegrates that result from an odd numbers of crossovers. In the mutS host, when the sequence homology of a pair of 405 bp substrates decreased from 100% to 89%, the recombinant frequency decreased by about 9-fold, while in the wild-type host the decrease was about 240-fold. These results suggest that multiple mismatches can reduce recombinant frequencies by impeding the mechanism of recombination itself, and by provoking mismatch repair. Single mismatches in 31 bp substrates caused reductions in recombinant frequencies of 2- or 12-fold, depending on the location of the mismatch. However, unlike the reduction by multiple mismatches, the reduction of the recombinant frequencies by single mismatches was the same in both mutS and wild-type hosts. Thus a single mismatch is sufficient to impede recombination, and mismatch repair seems unable to act on single mismatches in very short homologies during recombination.

Bursectomy in ovo blocks the generation of immunoglobulin diversity.

Immunoglobulins made by chickens bursectomized in ovo on day 11 of incubation were studied by two-dimensional gel electrophoresis. All such bursectomized chickens have limited diversity in their immunoglobulin molecules. A range of different degrees of diversity restriction was found in individual bursectomized chickens. The limited immunoglobulin diversity was stable for at least 20 weeks in that the same spots were found over this time span. Bursectomized chickens that responded to repeated antigen challenge had sheep erythrocyte- and dinitrophenol-specific antibodies of limited diversity. Different bursectomized responders made almost identical antigen-specific antibodies. The results were interpreted as indicating that individual bursectomized birds had been blocked at different points during the developmental generation of immunoglobulin diversity, and that the genetic mechanisms that underlie the developmental diversification of clones of B cells lead to a sequential generation of very similar antibody molecules in different embryos.

Sequence requirements for Sindbis virus subgenomic mRNA promoter function in cultured cells.

The Sindbis virus minimal subgenomic mRNA promoter (spanning positions -19 to +5 relative to the subgenomic mRNA start site) is approximately three- to sixfold less active than the fully active -98 to +14 promoter region. We identified two elements flanking the -19 to +5 region which increase its transcription to levels comparable to the -98 to +14 region. These elements span positions -40 to -20 and +6 to +14 and act synergistically to enhance transcription. Nine different virus libraries were constructed containing blocks of five randomized nucleotides at various positions in the -40 to +14 region. On passaging these libraries in mosquito cells, a small subset of the viruses came to dominate the population. Sequence analysis at the population level and for individual clones revealed that in general, wild-type bases were preferred for positions -15 to +5 of the minimal promoter. Base mutagenesis experiments indicated that the selection of wild-type bases in this region was primarily due to requirements for subgenomic mRNA transcription. Outside of the minimal promoter, the -35 to -29 region contained four positions which also preferred wildtype bases. However, the remaining positions generally preferred non-wild-type bases. On passaging of the virus libraries on hamster cells, the -15 to +5 region again preferred the wild-type base but most of the remaining positions exhibited almost no base preference. The promoter thus consists of an essential central region from -15 to +5 and discrete flanking sites that render it fully active, depending on the host environment.

Promoter for Sindbis virus RNA-dependent subgenomic RNA transcription.

Sindbis virus is a positive-strand RNA enveloped virus, a member of the Alphavirus genus of the Togaviridae family. Two species of mRNA are synthesized in cells infected with Sindbis virus; one, the 49S RNA, is the genomic RNA; the other, the 26S RNA, is a subgenomic RNA that is identical in sequence to the 3' one-third of the genomic RNA. Ou et al. (J.-H. Ou, C. M. Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982) identified a highly conserved region 19 nucleotides upstream and 2 nucleotides downstream from the start of the 26S RNA and proposed that in the negative-strand template, these nucleotides compose the promoter for directing the synthesis of the subgenomic RNA. Defective interfering (DI) RNAs of Sindbis virus were used to test this proposal. A 227-nucleotide sequence encompassing 98 nucleotides upstream and 117 nucleotides downstream from the start site of the Sindbis virus subgenomic RNA was inserted into a DI genome. The DI RNA containing the insert was replicated and packaged in the presence of helper virus, and cells infected with these DI particles produced a subgenomic RNA of the size and sequence expected if the promoter was functional. The initiating nucleotide was identical to that used for Sindbis virus subgenomic mRNA synthesis. Deletion analysis showed that the minimal region required to detect transcription of a subgenomic RNA from the negative-strand template of a DI RNA was 18 or 19 nucleotides upstream and 5 nucleotides downstream from the start of the subgenomic RNA.

Tn5supF, a 264-base-pair transposon derived from Tn5 for insertion mutagenesis and sequencing DNAs cloned in phage lambda.

We constructed a derivative of transposon Tn5 called Tn5supF for insertion mutagenesis and sequencing DNAs cloned in phage lambda. This element carries a supF amber-suppressor tRNA gene. Its insertion into lambda can be selected by plaque formation by using nonsuppressing (sup0) Escherichia coli for amber mutant lambda phage and sup0 dnaB-amber E. coli for nonamber lambda phage. Tn5supF is just 264 base pairs long. It transposes efficiently and inserts quasi-randomly into DNA targets. The unique sequences near its termini can be used as primer binding sites for dideoxynucleotide DNA sequencing, thus permitting the direct sequencing of DNAs cloned in phage lambda without subcloning.

The area-code hypothesis: the immune system provides clues to understanding the genetic and molecular basis of cell recognition during development.

Numberous studies of embryogenesis have provided evidence for highly specific cell-surface recognition phenomena. These include both the interactions of neighboring cells and the specific cellular migrations which occur as the developmental program of the embryo progresses. The area-code hypothesis elaborate here is an attempt to provide a framework for understanding cell-recognition phenomena in development. This hypothesis is based on extensive genetic, molecular, and cellular studies of the immune system. These studies suggest that the following events occur during the differentiation of antibody-producing cells. 1) Somatic cell lines of antibody-producing cells undergo a modification of their DNA as they become committed to synthesize a particular type of antibody molecule. This chromosomal modification event is probably a DNA translocation which leads to a somatic rearrangement of certain antibody genes. 2) In each of the specific cell lineages the new arrangement of DNA is inherited by all subsequent generations of cells. 3) The developmental programs which control these genetic alterations may be employed in a programmed and reproducible fashion. This programming of antibody development is suggested because different embryos appear to become committed to the production of identical antibody molecules in the same developmental sequence. 4) Antibody molecules are initially displayed on the cell surface where they serve as highly specifici receptors to trigger the cell to proliferate and differentiate upon interacting with appropriate external molecular signals. 5) Antibody-producing cells display combinations of different molecules on their surfaces which cause each of a very large number of different cells to interact differently with their environment. 6) The genes which code for many of these cell-surface molecules are organized into multigene families. These observations as well as information from other developmental systems have led us to propose the area-code hypothesis. This hypothesis is concerned with the structure, function, and regulation of cell-surface molecules that mediate recognition phenomena during embryogenesis. Area-code molecules are cell-surface molecules which are involved in the specific recognition phenomena during growth and development. These molecules provide cells with distinct cell-surface addresses or phenotypes, and provide the basis for the specificity in cell-cell recognition during cell migrations and cell-cell interactions, as well as serving as receptors for diffusible differentiation signals. The area-code hypothesis has 3 main postulates. i) There is a progressive display of specific combinations of area-code molecules on the surfaces of cells during development. ii) The genetic programs which determine the specific expression of area-code molecules are in part controlled by DNA modifications. These chromosomal modifications are believed to channel cells into specific lineages uith progressively restricted developmental options...