Showing papers by "David Baltimore published in 1984"

Ordered rearrangement of immunoglobulin heavy chain variable region segments.

Abstract: The immunoglobulin heavy chain variable region is encoded as three separate libraries of elements in germ-line DNA: VH, D and JH. To examine the order and regulation of their joining, we have developed assays that distinguish their various combinations and have used the assays to study tumor cell analogs of B-lymphoid cells as well as normal B-lymphoid cells. Abelson murine leukemia virus (A-MuLV) transformed fetal liver cells - the most primitive B-lymphoid cell analog available for analysis - generally had DJH rearrangements at both JH loci. These lines continued DNA rearrangement in culture, in most cases by joining a VH gene segment to an existing DJH complex with the concomitant deletion of intervening DNA sequences. None of these lines or their progeny showed evidence of VHD or DD rearrangements. Heavy chain-producing tumor lines, representing more mature stages of the B-cell pathway, and normal B-lymphocytes had either two VHDJH rearrangements or a VHDJH plus a DJH rearrangement at their two heavy chain loci; they also showed no evidence of VHD or DD rearrangements. These results support an ordered mechanism of variable gene assembly during B-cell differentiation in which D-to-JH rearrangements generally occur first and on both chromosomes followed by VH-to-DJH rearrangements, with both types of joining processes occurring by intrachromosomal deletion. The high percentage of JH alleles remaining in the DJH configuration in heavy chain-producing lines and, especially, in normal B-lymphocytes supports a regulated mechanism of heavy chain allelic exclusion in which a VHDJH rearrangement, if productive, prevents an additional VH-to-DJH rearrangement.

Preferential utilization of the most JH-proximal VH gene segments in pre-B-cell lines.

Abstract: The most JH-proximal VH gene segments are used highly preferentially to form VHDJH rearrangements in pre-B-cell lines. This result demonstrates that the rate at which immunoglobulin VH gene segments recombine is influenced by their chromosomal organization, and that the initial repertoire ofVH genes expressed in pre-B cells is strikingly different from that seen in mature populations.

Insertion of N regions into heavy-chain genes is correlated with expression of terminal deoxytransferase in B cells

Abstract: The variable regions of immunoglobulin heavy chains are encoded in the germ line by three discrete DNA segments: VH (variable) elements, D (diversity) elements and JH (joining) elements. During the differentiation of B lymphocytes, individual segments from each group are brought together by recombination to form the complete VHDJH variable region1–8. To understand these processes better, we have now isolated and sequenced molecular clones representing intermediates (DJH fusions) and final products ( VH-to-DJH joins) of heavy-chain gene rearrangement in two cell lines9,10 that represent analogues of cells at early stages of B-lymphocyte differentiation11–13. Heavy-chain gene assembly in one cell line but not in the other is accompanied by the appearance of short nucleotide insertions at the recombinational junctions. The generation of such insertions is positively correlated with the expression of terminal deoxynucleotidyl transferase in these lines.

Two regulatory elements for immunoglobulin kappa light chain gene expression

Abstract: By using internal deletions within a rearranged immunoglobulin kappa light chain gene, the presence of an intron regulatory sequence (enhancer) has been confirmed. Its presence is required for high-level transcription from a plasmid after transfection into myeloma cells. Transfection efficiency was monitored by the activity of a deleted H4 histone gene included in the plasmid. The intron element could be moved upstream of the gene in both orientations, fulfilling the definition of an enhancer. By using 5' deletions, a second regulatory element was located upstream of the "TATA" box, between positions -69 and -104. These two elements both are required for efficient kappa chain gene expression.

Joining of V K to J K gene segments in a retroviral vector introduced into lymphoid cells

Abstract: VK to JK recombination occurred within a DNA construct introduced into cells in the form of a defective retrovirus. Analysis of one recombinant demonstrated that VK–JK joining can occur via inversion with a net loss of a few base pairs.

Mechanism of RNA primer removal by the RNase H activity of avian myeloblastosis virus reverse transcriptase.

Abstract: The single-stranded DNA containing the Moloney murine leukemia virus origin for plus-strand synthesis was cloned in M13mp2 and used as a template for avian myeloblastosis virus reverse transcriptase in the presence of Moloney RNA which had been treated with pancreatic RNase A. The RNA pieces containing the polypurine stretch near the plus-strand origin were processed, presumably by RNase H, to generate primers for DNA synthesis which initiated both at the correct origin site and at one nucleotide downstream from the correct site. Approximately 50% of the labeled DNA fragments synthesized under these conditions retained the priming RNA on their 5' ends. When the isolated fragments were hybridized back to the template DNA and again treated with the reverse transcriptase, all of the RNA was removed from the labeled DNA. By using 5'-end-labeled pancreatic RNase A-resistant fragments, it was possible to show that the RNA primers were removed intact. It appears from these results that the RNase H activity associated with the enzyme shows a preference for cutting at the junction between the RNA and DNA moieties of such complexes and therefore is ideally suited for removing RNA primers.

Cloning of terminal transferase cDNA by antibody screening.

Abstract: A cDNA library was prepared from a terminal deoxynucleotidyltransferase-containing thymoma in the lambda phage vector lambda gt11. By screening plaques with anti-terminal transferase antibody, positive clones were identified of which some had beta-galactosidase-cDNA fusion proteins identifiable after electrophoretic fractionation by immunoblotting with anti-terminal transferase antibody. The predominant class of cross-hybridizing clones was determined to represent cDNA for terminal transferase by showing that one representative clone hybridized to a 2200-nucleotide mRNA in close-matched enzyme-positive but not to enzyme-negative cells and that the cDNA selected a mRNA that translated to give a protein of the size and antigenic characteristics of terminal transferase. Only a small amount of genomic DNA hybridized to the longest available clone, indicating that the sequence is virtually unique in the mouse genome.

Double-stranded cleavage by cell extracts near recombinational signal sequences of immunoglobulin genes.

Abstract: The genes encoding the variable regions of murine immunoglobulin light chains are present in the germ line in two separate segments, V and J. During B lymphocyte differentiation these segments are brought together to form a single unit (for review see ref. 1). Although much is known about the structures of V and J segments, both in germ-line configuration and after rearrangement, essentially nothing is known about the biochemical mechanism of V-J recombination. One possible step in proposed mechanisms of immunoglobulin gene rearrangement is endonucleolytic cleavage of the participating DNA segments before joining. In an attempt to detect such an activity, we have developed an assay for the detection of site-specific double- or single-strand endonucleolytic activity in crude soluble extracts. Using this assay we have detected an activity in extracts of nuclei from mouse B-lymphoid lines and from mouse L cells that is capable of introducing duplex breaks near the recombinational signal sequences of immunoglobulin JK segments. We report the activity here because of its intrinsic interest although we lack any direct evidence that it has a role in V-J recombination.

Liposome encapsulation of retrovirus allows efficient superinfection of resistant cell lines.

Abstract: Cell lines which are infected with retrovirus are resistant to superinfection by a related retrovirus. Packaging of whole virions within synthetic lipid vesicles allows efficient infection of such resistant cell lines. This system is more efficient in introducing encapsulated virus into infected cells than into uninfected cells.

Molecular basis of heavy-chain class switching and switch region deletion in an Abelson virus-transformed cell line.

Abstract: We demonstrated that a subclone of an Abelson murine leukemia virus-transformed B-lymphoid cell line switched from mu to gamma 2b expression in vitro, by the classical recombination-deletion mechanism. In this line, the expressed VHDJH region and the C gamma 2b constant region gene were juxtaposed by a recombination event which linked the highly repetitive portions of the S mu and S gama 2b regions and resulted in the loss of the C mu gene from the intervening region. An additional recombination event in this subclone involved an internal deletion in the S mu region of the expressed (switched) allele. One end of this deletion occurred very close to the switch recombination point. Despite the recombination-deletion mechanism of switching, the gamma 2b-producing line retained two copies of the C mu gene and two copies of the sequence just 5' to the S gamma 2b recombination point. The possible significance of the retention of these sequences to the mechanism of class switching is discussed.

Sexual preference of apparent gene conversion events in MHC genes of mice

Abstract: Polymorphisms exist at many genetic loci. At some loci, however, polymorphism is so high that tens and even hundreds of different alleles coexist in the population. Two such highly polymorphic systems are the immunoglobulin genes and the vertebrate major histocompatibility loci. The origin and maintenance of highly polymorphic loci remain open to debate but it seems likely that special mechanisms contribute to their variability and that their polymorphism serves important biological roles. The high degree of polymorphism at the H–2 class I major histocompatibility locus of the mouse has been documented by both tissue transplantation and serological methods1. More recently, molecular cloning and DNA sequencing of some of the class I genes has shown that most of the sequence variability is concentrated in the first two domains and is often found in clustered regions within them2–4. In addition, several groups have suggested that gene conversion events among the many class I genes may contribute to H–2 polymorphism5,6; such events would have to occur during meiosis to produce heritable alterations. The strongest evidence for gene conversion comes from sequence analysis of mutant class I H–2 alleles where concerted changes at adjoining sites in DNA imply gene conversion by distant but closely related loci5,6. We report here an analysis of these mutants indicating that the chromosomes containing loci that have experienced gene conversion originated from females. These data suggest a striking preference for mammalian meiotic gene conversion events during female rather than male gametogenesis.

PRODUCTION OF cDNA REPRESENTING HEPATITIS A VIRAL SEQUENCES

Abstract: Methods for producing HAV cDNA, products thereof, and uses thereof. HAV cDNA is produced, for example, by reverse transcribing HAV RNA and subsequently inserting the HAV cDNA into bacterial plasmids by genetic-engineering techniques. Transformed bacteria are then cloned and cultured to produce replicated chimeric plasmids containing the HAV cDNA. Such HAV cDNA is useful in assaying for the presence of HAV and in the production of HAV antigen and in the production of antibodies against HAV.

Molecular genetics of poliovirus.

Abstract: Poliovirus research has reached the point where molecular biologists and those interested in control of the disease can profitably come together. New molecular approaches offer the opportunity to make vaccines by previously inconceivable routes. These include synthesis of antigenic proteins in bacteria and chemical synthesis of antigenic peptides.