Andrew Becker

Mutations in the RB1 gene and their effects on transcription.

Inactivation of both alleles of the RB1 gene during normal retinal development initiates the formation of a retinoblastoma (RB) tumor. To identify the mutations which inactivate RB1, 21 RB tumors isolated from 19 patients were analyzed with the polymerase chain reaction or an RNase protection assay or both. Mutations were identified in 13 of 21 RB tumors; in 8 tumors, the precise errors in nucleotide sequence were characterized. Each of four germ line mutations involved a small deletion or duplication, while three somatic mutations were point mutations leading to splice alterations and loss of an exon from the mature RB1 mRNA. We were unable to detect expression of the mutant allele in lymphoblasts of three bilaterally affected patients, although the mutation was present in the genomic DNA and transcripts containing the mutations were obvious in the RB tumors in the absence of a normal RB1 allele. The variations in the level of expression of mutant transcripts suggest deregulation of RB1 transcription in the absence of a functional RB1 gene product.

Mechanisms of loss of heterozygosity in retinoblastoma

Retinoblastoma (RB) tumors arise when both alleles of the RB1 gene are inactivated by two mutational events (M1 and M2). M1 can be an initial germline or somatic mutation; M2 is frequently loss of heterozygosity (LOH), which makes the cell homozygous or hemizygous for the original mutation. LOH is the major mechanism by which many cancers are initiated. To further delineate the mechanism of LOH, we screened a total of 37 RB tumors for LOH by Southern blot analysis. The tumors were from 17 bilaterally and 17 unilaterally affected patients. Nineteen of 30 informative tumors (63%) from 27 patients showed LOH. Proximal and distal flanking markers on chromosome 13 were informative in 13 tumors, allowing evaluation of the mechanisms by which LOH occurred. Mitotic recombination was implicated in 6 (46%) of the 13 tumors.

DNA Packaging and Cutting

INTRODUCTION The head of bacteriophage λ is assembled when the products of two separate synthetic pathways come together. One, the DNA replication pathway, produces a linear polymer of λ chromosomes called a concatemer. In the other pathway, λ head proteins assemble to produce the empty capsid shell known as the prohead (Georgopoulos et al., this volume). DNA and proheads interact, and a chromosome from the concatemer is inserted into the cavity of the prohead and is separated from the unencapsidated remainder of the DNA by endonucleolytic cutting. Knowledge of λ head assembly was presented by Yarmolinsky (1971) and by Kellenberger and Edgar (1971) in The Bacteriophage Lambda . The review of Hohn and Katsura (1977) offers a more recent summary of the subject. Casjens and King (1975), Murialdo and Becker (1978b), and Wood and King (1979) have written comparative reviews on head morphogenesis and DNA packaging. Most recently, the problem of DNA packaging in the double-stranded DNA phages has been updated in an elegant synthesis by Earnshaw and Casjens (1980). Because of space limitations in this paper, we restrict discussion of other viruses to results not emphasized in other reviews. CHROMOSOME STRUCTURE AND DNA PACKAGING The linear λ chromosome is injected into the cell, and annealing and ligation of the cohesive ends rapidly cyclize the molecule. Early in the growth cycle, bidirectional replication results in progeny rings. Later, rolling-circle replication produces the concatemers that are the substrates for DNA packaging (Skalka 1977; Furth and Wickner, this volume). DNA packaging generates mature...

Mechanism of cos DNA cleavage by bacteriophage λ terminase: Multiple roles of ATP

In the terminus-generating (ter) reaction of phage lambda, the phage enzyme terminase catalyzes the production of staggered nicks within the cohesive-end nicking site (cosN). Although the two nicks are related by a rotational symmetry axis that bisects cosN, the in vitro ter reaction is strikingly asymmetric at the nucleotide level. Nicking of the lambda r strand precedes nicking of the I strand. Furthermore, when the two nicking reactions are uncoupled, they have different nucleotide cofactor requirements. ATP plays critical roles during cos cleavage: First, nicking of both DNA strands is stimulated by the addition of ATP. Second, ATP is required for the correct specificity of r-strand nicking since, in the absence of nucleotide, the r-strand nick is shifted 8 bases to the left. Studies with nonhydrolyzable analogs indicate that ATP hydrolysis is not required for these functions. However, after the two nicks are made, terminase catalyzes a disengagement of the cohered ends in a reaction that requires ATP hydrolysis.

Studies on an in vitro system for the packaging and maturation of phage λ DNA

Abstract Packaging and maturation of λ DNA in cell-free extracts is arbitrarily partitioned into two stages. In stage 1, exogenously added, immature concatemeric λ DNA interacts with the A-gene product (pA) and phage proheads. After this reaction has occurred, a second extract is added to initiate a different set of events that results in phage production (stage 2). The use of highly purified pA and purified proheads during stage 1 results in a marked reduction in the final phage yield. The deficit is largely corrected by the addition of extracts of uninfected cells in stage 1. This observation suggests that one or more host factors are required in the packaging reaction. The testing in vitro for λ-specific requirements in stage 1 reveals that Nu1-negative extracts prepared from λt16-infected cells are phenotypically prohead positive, but pA negative, in the assay. This result indicates that the t16 mutation is in a locus that specifies a gene product. Complementation in vitro between λt16- and λAam-infected cell extracts has not been accomplished as yet. Paradoxically, highly purified pA can support optimal phage production in conjunction with Nu1-negative stage 1 and stage 2 extracts. Stage 2 requires the action of the products of λ genes D, W, and FII. Successful complementation among pairs of stage 2-deficient extracts provides convenient assays for purifying these proteins.

Prediction of an ATP reactive center in the small subunit, gpNu1, of the phage lambda terminase enzyme

The small subunit of the bacteriophage lambda terminase enzyme, the product of the phages Nu1 gene, is shown to contain amino acid segments homologous to those present in a large number of ATPases. In keeping with these predictions, the purified protein has been found to hydrolyze ATP with a relatively low turnover number. Terminase holoenzyme is a known ATPase, and the biochemical significance of an ATP-interactive center situated in the gpNu1 subunit is discussed.

Bacteriophage lambda DNA maturation. The functional relationships among the products of genes Nul, A and FI.

Abstract The FI gene of bacteriophage λ functions in head assembly, but its exact role is not well understood. FI mutants are leaky, producing between 0.1 and 0.5 viable particles per infected cell. In order to investigate the function of the FI product (gpFI) in vivo , mutants of λ were isolated that are able to grow in the absence of gpFI. These mutants, called fin (for FI independence) map in the region of gene Nul and the beginning of gene A . Proteins made in cells infected with the fin mutants were labelled with [ 35 S]methionine and analysed by polyacrylamide gel electrophoresis. In addition, the levels of activity of the A product were measured in the in vitro DNA packaging assay. As a result of these experiments, the fin mutants can be classified in two groups. Upon infection, fin mutants of one group selectively produce three to fivefold more gpA than do wild-type phage fin mutants of the second group do not overproduce any λ late gene product detectable by the autoradiographic technique. gpA overproducers can also be isolated by selecting for λ Aam Wam phages that can plate on a weak suII cell strain. The mutation responsible for this pseudoreversion is called A op and maps in the Nu1-A region. A op is also a fin mutation, since its presence in λ FI − enables it to plate on non-permissive hosts. Therefore, it seems that one condition sufficient for normal growth of FI − phage is the overproduction of gpA. The nature of the fin mutations that do not result in gpA overproduction is discussed.

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.

Late stages in bacteriophage λ head morphogenesis: In vitro studies on the action of the bacteriophage λ D-gene and W-gene products

Abstract The in vitro maturation of bacteriophage λ can be divided into discrete steps. Concatemers of λ DNA bind terminase to form complex I. This DNA-terminase complex then binds a prohead to form a ternary complex (II). Complex II in turn can be converted to infectious phage by the addition of extracts containing the products of the phage genes D, W, FII , as well as phage tails. By using in vitro complementation assays gpD and gpW have been partially purified and their interactions with complex II studied. gpD can bind to complex II in vitro to form a new complex (III) which can be isolated by sedimentation on neutral sucrose gradients. This complex requires only the addition of gpW, gpFll, and phage tails to form mature phage particles. The sedimentation of complex III is virtually identical to that of complex II; however, the resistance of the former to inactivation by DNase is higher, likely due to the partial packaging of the DNA. In similar experiments it was shown that gpW cannot bind to complex II but can effectively interact with complex III. This latter reaction converts complex Ill to a DNase-resistant form which sediments in a manner identical to that of full phage heads (complex IV). After isolation of the complex IV only gpFll and tails are required for mature phage formation in vitro . gpW is a heat-stable protein of molecular weight approximately 10,000.

A Genetic analysis of bacteriophage lambda prohead assembly in vitro

Abstract Phage lambda DNA can be packaged into proheads to generate full heads in vitro . Upon addition of tails and other gene products to the cell-free assembly system, infectious phage are formed. This system provides a convenient assay to measure biologically active proheads. Thus, it has been shown that biologically active proheads are not produced in wild-type cells infected with phage mutants in genes B , C , Nu3 and E , nor in groE mutant cells infected with wild-type phage. However, proheads can be assembled in vitro upon incubation of a mixture of λ Nu 3 − and λ E − infected-cell extracts. This complementation reaction has been analyzed using extracts prepared from wild-type or groE − cells infected with phage carrying amber mutations in one or two prohead genes. The conclusion from this analysis can be summarized as follows. 1. (1) Among all the possible combinations of extracts of cells infected with single amber mutants, the E − + Nu 3 − combination is the only one that gives substantial complementation in vitro . 2. (2) gpC † can be contributed by either member of the complementing pair of extracts. 3. (3) An E − B − extract will not complement a Nu 3 − extract. 4. (4) Active gpB can only be derived from a Nu 3 + extract, and/or active gpNu3 can only be derived from a B + extract. It is, therefore, probable that gpB and gpNu3 interact in vivo before gpE is polymerized. 5. (5) The lack of in vitro complementation exhibited by certain pairs of mutant extracts can be explained conveniently by postulating that essential gene products (such as gpE) are consumed in the assembly of abnormal capsid structures in vivo . 6. (6) The B − Nu 3 − extract (gpE and gpC donor) does not require the function of the host groE gene product to provide complementing activity. 7. (7) groE function is essential for the production of the complementing activity in E − C − extracts (gpB and gpNu3 donor). 8. (8) If gpB and gpNu3 are synthesized in the absence of wild-type groE function, the defect in complementing activity cannot be repaired by the addition of groE + cell extracts. It thus appears that groE functions very early during prohead assembly, prior to the polymerization of the coat protein gpE.