HIV-1 indicator cell lines.

A simple quantitative bioassay for infectious HIV-1 has been developed. The assay is based on adherent CD4+ HeLa cell lines stably transfected with episomal vectors carrying the Escherichia coli beta-galactosidase gene under the control of the HIV long terminal repeat (LTR) promoter. HIV infection of these cell lines transactivates the LTR promoter inducing beta-galactosidase production. Infected cells and virus foci can be stained dark blue by the addition of the chromogenic substrate X-Gal. Alternatively, a readily automatable quantitative enzyme assay can be performed on the infected cultures. Because of its simplicity the bioassay may be useful for routine quantification of HIV-infected cultures, plaque purification, virus neutralization studies and for the screening of antiviral agents.

Syncytia--a major site for the production of the human immunodeficiency virus?

An HIV-1 isolate (designated GB8) was isolated from an asymptomatic AIDS patient and subsequently grown in the T-lymphocyte cell line JM. Electron microscopy showed that it is vesicles within the syncytia present in cultures of JM cells chronically infected with GB8, rather than the surface membranes of unfused cells, which are the major sites for the assembly, production and release of HIV.

Human cytomegalovirus DNA: BamHI, EcoRI and PstI restriction endonuclease cleavage maps.

The cloned HindIII fragments of human cytomegalovirus (HCMV) strain AD169 DNA were mapped with respect to the BamHI, EcoRI and PstI restriction endonuclease cleavage sites. Composite restriction endonuclease cleavage maps for the entire virus genome were constructed using the previously established linkages between the HindIII fragments.

Nucleotide sequence of the most abundantly transcribed early gene of human cytomegalovirus strain AD169.

Cytoplasmic poly(A+)RNA was isolated from human embryo fibroblast cells during the early phase of an infection with human cytomegalovirus strain AD169. These preparations contained a single abundant transcript of 2.7 kb which was derived from each of the repeat sequences flanking the long unique region of the virus genome. The gene was unspliced and poly(A+)RNA derived from it continued to accumulate in the cytoplasm of cells during the later stages of infection. This gene and the surrounding region was sequenced. A potential open reading frame of 170 amino acids was identified close to the 5'-terminus of the gene; the 2.7 kb early transcript therefore contains a long untranslated 3'-sequence. The promoter sequences of the 2.7 kb early gene show homology with corresponding regions of the HSV-1 gD and the human hsp 70 genes.

Transcription of the immediate early genes of human cytomegalovirus strain AD169.

Cloned sub-genomic fragments of human cytomegalovirus strain AD169 were used to analyse immediate-early (IE) transcription in virus-infected cells. Transcriptionally active regions of the HCMV genome were identified by hybridising cytoplasmic IE poly(A)+-RNA with dot blots and Southern transfers of restriction endonuclease digests of recombinant plasmids. The size, number and, in some cases, the orientation of transcription of IE RNA species were determined. The most abundant IE mRNA (IE-1.95) was mapped at 0.0764-0.0865 map units. The transcription of two middle abundant (1.7 and 2.15 kb) IE RNAs was initiated immediately downstream, and in the same orientation as the IE-1.95 gene. A second transcriptionally active area was identified at 0.593-0.619 map units. Three mRNA species (IE-1.75, IE-3.8 and IE-4.8) were derived from this region. Additional minor IE transcription was also observed from other regions of the HCMV genome. Hybrid-selected translation was used to identify the polypeptides encoded by the major IE RNA species.

The detection of subtle differences between different orthopoxvirus genomes by heteroduplex analysis.

Corresponding DNA fragments from variola (Harvey) and monkeypox (Denmark) viruses which had been cloned into different plasmid vectors were subjected to heteroduplex analysis. Characteristic deletion loops corresponding to differences between the cloning vectors served as internal markers to identify and to orientate the heteroduplexed molecules. Partial denaturation of the resulting heteroduplexes was used as a primary screen to locate regions of heterogeneity between the poxvirus inserts. The denaturation threshold for homoduplexes was consistently higher than that for heteroduplexes. However, significant sequence divergence between corresponding fragments was indicated by larger than usual differences in thresholds between corresponding homo- and heteroduplexes. Denaturation bubbles of 0.1-0.5 kb were detected and hence small regions of heterogeneity between the genomes (180 kb) of variola and monkeypox viruses were localised. This procedure has a general application in comparative studies on large, complex but closely related DNA molecules.

Studies on the specificity of the vaccine effect elicited by inactivated simian immunodeficiency virus.

Inactivated, partially purified simian immunodeficiency virus (SIVmac) protected macaques from intravenous challenge with homologous and heterologous strains of SIV that had been grown on human cells but no protection against challenge with monkey peripheral blood mononuclear cell-grown SIVmac was afforded. Human immunodeficiency virus type 1 prepared in an analogous way to the SIVmac vaccine on the C8166 human T cell line protected macaques against challenge with human cell-grown SIVmac. These results suggest that protection may be mediated by xenoimmunization with the vaccine cell substrate proteins. All vaccinated macaques had anti-cell antibodies. Major reactivity to MHC class I antigens was found as well as to a 70-kD protein detectable only under nonreducing conditions.

Detection of cytomegalovirus by DNA-DNA hybridization employing probes labelled with 32-phosphorus or biotin.

Various factors influencing the detection of human cytomegalovirus (HCMV) in infected cells by DNA-DNA hybridization have been investigated. Employing the Hind III O fragment of HCMV AD169 labelled with 32P, we found that detection sensitivity was highly influenced by the method employed for extraction of DNA from infected cells. Excision of the Hind III O fragment from the vector by restriction endonuclease digestion prior to 32P-labelling further improved the detection capability of the probe. Similarly, cytomegalovirus (CMV) DNA detection employing biotin-labelled probes and streptavidin/alkaline phosphatase in the hybridot assay was also highly dependent on the method of DNA extraction prior to hybridization. Finally, we describe an in situ assay employing a biotin-labelled probe and fluorescein-conjugated avidin to detect CMV DNA in cultured cells.

Urate deposits in the gecarcinid land crab Gecarcoidea natalis are synthesised de novo from excess dietary nitrogen

The urate content of the gecarcinid land crab Gecarcoidea natalis was correlated to the amount of nitrogen assimilated. Crabs fed a high-nitrogen diet (ad libitum amounts of soy beans and fig leaves, Ficus macrophylla) for 6 weeks assimilated approximately 23 times more nitrogen (33.9±5.6mmolkg-1day-1) than animals fed a diet low in nitrogen (fig leaves alone) (1.5±0.7mmolkg-1day-1). Animals maintained on a high-nitrogen diet accumulated urate (67.1±29.4mmolkg-1drymass), while animals fed the low-nitrogen diet did not accumulate significant amounts of urate compared with the control animals killed at the beginning of the dietary period. The urate deposits clearly originate from the excess dietary nitrogen ingested on the high-nitrogen diet. The intake of preformed dietary purine was low (0.028±0.005mmolkg-1drymass) and at most could only account for 0.04% of the urate accumulated by crabs fed the high-nitrogen diet. This indicates that the urate was synthesised de novo. When crabs were fed a high-nitrogen diet supplemented with [15N]glycine, the 15N heavy isotope was incorporated into urate. This provided direct evidence that the urate was synthesised de novo.

GAS EXCHANGE THROUGH THE LUNGS AND GILLS IN AIR-BREATHING CRABS

Lung and gill performance in gas exchange have been evaluated in eight species of air-breathing crabs with two different lung circulatory designs, those with portal systems and smooth lung linings, and those without portal systems and with invaginated and evaginated lung linings. In all species, the lungs were extremely effective in oxygen uptake whilst the performance of the gills was inferior. An exception to this was Gecarcoidea natalis, which has gills highly modified for aerial gas exchange; its gills and lungs were equally efficient in O2 uptake. The relative efficiencies of the lungs and gills in CO2 excretion differed between species, with the gills being the major site of CO2 loss in the more amphibious species and the lungs having an increasingly important role in the more terrestrial crabs. The presence or absence of lung portal systems was not found to correlate with either saturation rates or efferent oxygen concentrations, with both lung types being extremely efficient in O2 uptake. The lungs with portal systems showed a large increase in oxygen content in the first lacunar bed and progressively smaller increases in the next two; these lungs may, therefore, have some reserve for exercise.

NITROGENOUS EXCRETION IN THE TERRESTRIAL CARNIVOROUS CRAB GEOGRAPSUS GRAYI: SITE AND MECHANISM OF EXCRETION

The rate and mechanism of nitrogen excretion were examined in Geograpsus grayi. This species excretes waste nitrogen as gaseous NH3 in periodic bursts. The mean concentration of total ammonia ([NH3]+[NH4+]) in the primary urine during bursts of excretion (1.72 mmol l-1) was similar to that of haemolymph (2.07 mmol l-1) but was significantly lower (P<0.005) than that of branchial fluid (80.6 mmol l-1). The effects of ion exchange inhibitors on the apical membrane of the gill epithelium in Geograpsus grayi were examined. The presence of an amiloride-sensitive Na+/NH4+ exchanger was confirmed and a SITS-sensitive Cl- influx suggested Cl-/HCO3- exchange. Thus, the site of nitrogenous excretion in this species is the branchial chamber, which is also the site of reprocessing of urine for ion regulation in other terrestrial crabs. Gaseous ammonia excretion is achieved by volatilisation of NH3 from the branchial fluid. High partial pressures of ammonia in the branchial fluid are produced by apical Na+/NH4+ exchange and elevation of the pH.

Plasmid DNA isolation (sheared lysate method).

The cleared lysate method (see Chapter 25 ) is not usually very effective for isolation of plasmids larger than about 20 kb. Recovery of plasmid DNA is often poor, presumably because high molecular weight plasmids are removed by the clearing spin. An alternative procedure, therefore, is to load the complete cell lysate onto a cesium chloride-ethidium bromide gradient that will separate the plasmid DNA from the chromosomal material and also from other cell components.

Small-scale plasmid DNA preparation.

For the initial characterization of a recombinant plasmid, it is necessary to determine the size of the plasmid or, preferably, the size and characteristics of the insert itself. A method is therefore required for the simultaneous preparation, from a number of isolates, of plasmid DNA in a state sufficiently pure for restriction enzyme digestion. The requirements of such a procedure are: (i) A simple method for rapid lysis of the bacterial cells. (ii) Separation of plasmid from chromosomal DNA. (iii) Removal of proteins and of other components of the cells that might interfere with restriction enzyme treatment. (iv) Removal of detergents, salts, etc. used in the process.

Preparation of Chromosomal DNA from E. coli.

This chapter describes a simple and rapid way of extracting and purifying chromosomal DNA from E. coli and many other species of bacteria. This procedure is essentially a simplified version of that described by Marmur in 1961 (1). The cells are lysed by treatment with a detergent and the mixture is deproteinized by phenol-chloroform extraction. Further purification can be achieved by treatment with ribonuclease and proteinase K. The resulting DNA, free of protein and RNA contamination, is sufficiently pure to be used for restriction digestion and cloning, e.g., in the preparation of gene libraries.

Preparation and assay of phage lambda.

Lambda, a temperate bacteriophage of E. coli, has two alternative modes of replication in sensitive cells, known as the lytic and lysogenic cycles. In the lytic cycle, after the lambda DNA enters the cells, various phage functions are expressed that result in the production of a large number of mature phage particles and cell lysis. In the lysogenic mode, which normally occurs in only a small proportion of the infected cells, the phage forms a more or less stable relationship with the host bacterium; this stable state is known as lysogeny. In a lysogenic cell, phage DNA is normally incorporated into the chromosomal DNA via specific attachment sites on both the phage DNA and the host chromosome. Replication of lambda DNA then occurs only during replication of the host chromosome, and the phage genome is inherited by each daughter cell at cell division. The phage is maintained in this prophage state through the action of a repressor protein, coded for by the phage gene cl. This repressor protein turns off the expression of virtually the whole of the lambda genome. If the repressor is inactivated, the expression of phage genes is initiated. This leads to the excision of lambda DNA from the host chromosome and entry into the lytic cycle. The balance between the lytic and lysogenic modes of replication is a delicate and complex one in which a key factor is the concentration of the cl gene product. Some of the many sources of further information about the basic biology of lambda phage are listed in the references to this chapter.

Preparation of phage lambda DNA.

The isolation of phage DNA from a purified phage preparation simply involves the removal of the proteins of the phage particle. This is done most easily by phenol extraction (Method A). The large amounts of high quality DNA needed for use as vectors or for markers for gel electrophoresis can be obtained in this way. However, sometimes (for example, when screening recombinant phages) only small amounts of phage DNA are required. The purity of these samples needs only to be sufficient for the restriction enzyme digests to provide distinct patterns on an agarose gel. Method B provides one way in which this can be done (1). A plate lysate is prepared and the released phage is subsequently lysed by the addition of SDS. The phage proteins and SDS are precipitated by the addition of potassium acetate to leave the phage DNA in solution. Finally, the DNA is recovered by ethanol precipitation.

The use of alkaline phosphatase to prevent vector regeneration.

Linearized plasmid vector molecules produced by restriction enzyme digestion can be reformed into circles by the action of DNA ligase. This is the most favorable reaction even when foreign DNA fragments are present. Thus, unless a direct selection technique is available, the reformed parental molecule will also give transformants and hence reduce the overall cloning efficiency. Usually most of the recircularized parental plasmids can readily be distinguished from recombinants by the absence of insertional inactivation; nevertheless, this may involve screening large numbers of transformants for each recombinant.

Bacterial transformation (kushner method).

The principle of this procedure is very similar to that of the standard calcium chloride method, (see Chapter 34 ) i.e., exponential phase cells are harvested and washed with a solution containing divalent cations. This renders the cells competent, which simply means they are now able to take up DNA from the solution. After mixing the competent cells with the DNA, they are subjected to a heat shock to promote the uptake of DNA, presumably by affecting the physical state of the lipids in the membrane.

In vitro packaging of DNA.

In the normal growth cycle of bacteriophage lambda, the proteins that ultimately form the head of the phage particle are assembled into an empty precursor of the head (prehead); the phage DNA is replicated separately and then inserted into the empty head particles-a process known as packaging. A number of phage gene products play an important role in this process. Amongst these are: (i) The E protein, which is the major component of the phage head; mutants that are defective in this gene are unable to assemble the preheads, and therefore accumulate the other, unassembled, components of the phage particle as well as the other proteins involved in packaging. (ii) The D and A proteins, which are involved in the packaging process itself. Mutants that are defective in these genes are able to produce the preheads, but will not package DNA. This results in the accumulation of empty preheads.

Identification of recombinant phages by plaque hybridization.

Phage vectors are often used rather than plasmids, particularly for the production of gene "banks" or "libraries." The plaques produced can be screened for the presence of specific DNA sequences by hybridization using a procedure similar to that used for colony hybridization (see Chapter 42 ). Plaque hybridization offers some advantages over colony hybridization, largely because the area of the filter to which the DNA is bound is smaller and more defined. A higher density of plaques can therefore be used, which in turn means that more plaques can be screened in a single hybridization-several thousand on an ordinary (8.5 cm) petri dish; using larger containers such as baking dishes or trays the number can run into hundreds of thousands in a single hybridization. In addition, since the location of the plaques is not disturbed by the process, nor are they smudged in the way that colonies can be, several replicates can be taken from the same plate. This means that the same set of plaques can be screened for hybridization to several different probes.