Biological containment could decrease the uncertainties surrounding the deliberate release of recombinant microorganisms

The chromosomal genes gef and relF from Escherichia coli and the plasmid-encoded genes hok, flmA, srnB, and pndA constitute the gef gene family, which encodes a cell-killing function. In order to investigate the mechanism of cell killing we have isolated an E. coli mutant strain that is resistant to the overexpression of the toxic proteins encoded by the gef gene family. This phenotype requires at least two mutations, one of which has been mapped to 55.2 minutes. This mutation was sequenced and shown to represent a single base substitution in an open reading frame (ORF178) encoding a putative membrane protein having a molecular mass of 20.1 kDa. ORF178 and an upstream frame, ORF190, probably constitute an operon.

Two hundred eleven HIV-seropositive patients with AIDS, AIDS-related complex, or a CD4+ cell count less than 200 x 10(6) were examined for the presence of hepatitis B virus markers during the course of their HIV infection (median follow-up of 18 months; range of 1 to 107 months). Anti-HBs was detected initially in 138 patients (65%). Sixteen patients (8%) were HBsAg positive at entry. Fourteen had chronic HBV infection of whom 12 initially were positive for HBeAg and HBV DNA; 11 remained positive during follow-up, whereas one seroconverted to anti-HBe and lost HBV DNA. Two patients with chronic HBV infection were initially negative for HBeAg and HBV DNA: one later had reactivated HBV replication and one cleared HBeAg following onset of hepatitis D infection. The last two HBsAg-positive patients had resolving acute HBV infection. Six of the 57 patients who initially were negative for HBV markers acquired HBV infection during follow-up. Four of these six patients developed chronic infection whereas two patients had acute subclinical resolving hepatitis. In addition, four patients became HBsAg positive with their last serum samples, possibly indicating reactivation of HBV infection following progressive immunological and clinical deterioration. None of the patients developed clinical symptoms that could be ascribed to HBV infection, and transaminase elevations were only sporadically recorded. It is concluded that acquisition of HBV infections is not infrequent in HIV-seropositive patients with immune deficiency. Furthermore, the course of both previously established chronic HBV infection and newly acquired HBV infection is modified in such patients, whereas reactivation of past HBV infection seems to be a rare event.

Reactivation of hepatitis B virus replication was investigated in an unselected group of 44 HBV DNA negative, anti-HBe positive chronic HBsAg carriers. Twenty-five patients (54%) were intravenous drug addicts and 7 (16%) were male homosexuals. Sixteen patients had evidence of delta infection and five of the seven male homosexuals had human immunodeficiency virus infection. The patients were followed for 1 to 180 months (median, 24 months) while HBV DNA negative, anti-HBe positive. Reactivation, defined as reappearance of HBV DNA or HBeAg, or both, was detected in six patients corresponding to an annual reactivation rate of 5%. Reactivation in four patients was detected by reversion to HBV DNA positivity only, whereas HBeAg/anti-HBe status remained unchanged. Two patients became both HBV DNA and HBeAg positive. None of the patients developed hepatitis-like symptoms and transaminase elevation was only observed in two patients. Reactivation in two patients was ascribed to human immunodeficiency virus infection and in one patient to chronic lymphatic leukaemia. It is concluded that HBV DNA seems to be superior to HBeAg in the detection of reactivation of HBV replication and that reactivation associated with clinical symptoms leading to progression in chronic liver disease is a rare event in the population studied.

The relF gene in Escherichia coli is related to the hok gene on plasmid R1. Both genes encode small proteins which, when overexpressed in E. coli lead to collapse of the membrane potential and cell death. A third gene, designated gef, which encodes a homologous cell-toxic protein, has been isolated from E. coli DNA. Both gef and relF are transcribed in E. coli and subject to post-transcriptional regulation which, in the case of gef, is coupled to translation of a leader sequence. The finding of homologous sequences in such distantly related bacteria as Agrobacterium and Rhizobium species suggests an important physiological role.

The presence of hepatitis B virus and delta agent markers was investigated in 41 patients referred during the years 1970-1985 with fulminant hepatitis classified as type B or non-A non-B and compared to findings in patients with uncomplicated hepatitis B and chronic hepatitis B infection. 13 patients had no markers of hepatitis B and delta infection and were classified as non-A non-B hepatitis. The remaining 28 patients were all HBsAg and IgM anti-HBc positive and 14 (50%) had evidence of delta infection. In contrast, only 13/71 patients (18%) with acute benign hepatitis B had evidence of delta coinfection (p less than 0.005). This corresponds to an odds ratio of 4.5 for development of fulminant hepatitis among patients with hepatitis B and delta coinfection. In 100 chronic HBsAg carriers 29% were positive for delta markers. 12 of the delta infected patients with fulminant hepatitis were positive for total antibody to the delta antigen, and 2 were delta antigen positive. Three were HBeAg positive/anti-HBe negative. None had hepatitis B virus DNA. Among the 14 patients without delta infection, hepatitis B virus DNA was found in 2/4 HBeAg positive/anti-HBe negative patients and in 1/8 patients negative for both markers. The present data indicate that a high proportion of Danish patients with fulminant hepatitis B have hepatitis B and delta agent coinfection. Further, the findings suggest that hepatitis B and delta coinfection may be associated with an increased risk of development of fulminant hepatitis as compared to that of hepatitis B alone.

The presence of antibodies to HTLV-III and markers of active hepatitis B virus replication was examined in a longitudinal study of 33 consecutive male homosexual HBsAg carriers. The mean follow-up time was 37 months (range = 4 to 109 months). All patients were initially hepatitis B virus DNA-positive and HBeAg positive. Antibodies to HTLV-III were detectable in eight patients while they were positive for both of these markers. One of them cleared hepatitis B virus DNA and seroconverted from HBeAg to anti-HBe. This corresponds to an annual clearance/seroconversion rate of 4% (95% confidence limits = 0 to 15%). In two patients, antibodies to HTLV-III appeared after clearance of hepatitis B virus DNA and HBeAg, and in one of them, hepatitis B virus DNA reappeared. Among the 25 patients negative for HTLV-III antibodies, the annual hepatitis B virus DNA clearance rate was 20% and HBeAg to anti-HBe seroconversion rate was 11% (95% confidence limits = 11 to 31% and 4 to 20% respectively). The observed hepatitis B virus DNA clearance rates in the two groups were significantly different (p less than 0.05). Disease activity, as determined by transaminase levels, was significantly lower in HTLV-III-infected individuals as compared to individuals without HTLV-III infection (p less than 0.05). Infection with HTLV-III may extend the period of active viral replication or even reactivate hepatitis B virus replication and seems to diminish inflammatory disease activity in chronic HBsAg carriers.

The presence of hepatitis B virus DNA and anti-δ was examined in a longitudinal study of 24 patients known to be δ-infected during the course from acute to chronic hepatitis B virus infection. Fifteen patients (63%) were hepatitis B virus DNA positive in the first serum sample. Eleven of 14 patients, who cleared hepatitis B virus DNA, did so following or at the same time as onset of δ-infection. Duration of hepatitis B virus DNA positivity in these 11 patients was shorter than in 11 anti-δ-negative controls matched according to duration of preceding hepatitis B virus DNA positivity, but the difference was not statistically significant. Considering only patients positive for IgM anti-δ in the last serum sample (eight patients), a statistically significant shorter duration of hepatitis B virus DNA positivity was found in δ-infected patients than in the controls (p < 0.02). The study indicates that the δ-agent may have the capacity to inhibit hepatitis B virus replication and that a chronic δ-infection may lead to a termination of the period of active viral replication.

The presence of hepatitis B virus (HBV) DNA was investigated in 26 hepatitis B surface antigen-positive blood donors. Three donors (12%) were concordantly positive for HBV DNA and hepatitis B e antigen (HBeAg) and had IgM antibody to hepatitis B core antigen (anti-HBc). Two donors (8%) had HBV DNA without HBeAg; both were positive for antibody to HBeAg and lacked IgM anti-HBc. Twenty-one HBV DNA-negative donors had antibody to HBeAg, and all were negative for HBeAg and IgM anti-HBc. Blood units from 16 donors were transfused. A sufficient serological and clinical follow-up was available for 10 HBV-susceptible recipients. Three recipients of HBV DNA-positive blood units were infected irrespective of HBeAg status or presence of IgM anti-HBc. Six (86%) of seven recipients of HBV DNA-negative blood units developed HBV infection. Thus all hepatitis B surface antigen-positive blood donors should still be considered infectious irrespective of status with regard to HBeAg, HBV DNA, and IgM anti-HBc.

Sera from 77 consecutive patients with acute type B hepatitis were examined for hepatitis B virus DNA (HBV DNA) by a spot hybridization method. The median follow-up time was 8 months (range, 1 week to 3 years). HBV DNA was detected in 26 (34%) patients on admission to the hospital. A significant positive correlation was found between short duration of symptoms and the presence of HBV DNA (p less than 0.025). Twenty-four (46%) of 52 HBeAg-positive patients were HBV DNA positive compared to 2 HBV DNA-positive patients of 25 HBeAg-negative patients (8%) (p less than 0.001). Four HBeAg-negative patients had serum HBV DNA initially or during follow-up; three had anti-HBe. Six of 77 patients with acute type B hepatitis (8%) became chronic HBsAg carriers, and HBV DNA was detectable from 5 months to more than 3 years after onset of symptoms. The presence of serum HBV DNA for more than 8 weeks after initial symptoms may predict development of a chronic HBsAg carrier state. In none of the chronic carriers was serum HBV DNA present after clearing of HBeAg.

The presence of hepatitis B virus DNA in serum was determined in 57 unselected patients during the course from acute to chronic hepatitis B infection. Forty-six (81%) patients were hepatitis B virus DNA-positive in the first available serum sample. Generally, hepatitis B virus DNA was cleared before or at the same time as HBeAg, but in two patients (4%), hepatitis B virus DNA could be demonstrated after HBeAg clearance. One of the latter patients had hepatitis B virus DNA in the presence of anti-HBe. Both patients became hepatitis B virus DNA-negative. Seven of the hepatitis B virus DNA-positive patients received long-term treatment with prednisone, and three of them continued to be hepatitis B virus DNA positive for more than 10 years. Among the untreated patients hepatitis B virus DNA could be detected for up to 7 years, and 10 patients were hepatitis B virus DNA-positive for three years or more. Twenty-four patients (42%) showed serological signs of delta agent infection. Hepatitis B virus DNA clearance was observed in a significantly higher proportion (87%) of delta-infected patients as compared to patients with no delta infection (45%) (p less than 0.05). In addition patients with delta infection had a significantly increased hepatitis B virus DNA clearance rate as compared to patients without delta markers in their serum (p less than 0.01). In one (8%) delta-infected patient, hepatitis B virus DNA clearance was followed by a fall in transaminases into the normal range as opposed to results in 86% of patients with pure hepatitis B (p less than 0.002).

The longest DNA molecules synthesized by endogenous reverse transcription in detergent-permeabilized Moloney murine sarcoma virus (Mo-MSV) virions (clone G8-124) are double-stranded DNA molecules of 5.8 kilobase pairs (kbp). This DNA species has been purified by sedimentation of total in vitro synthesized Mo-MSV DNA through neutral sucrose gradients. A physical map of the positions of the cleavage sites for a series of restriction endonucleases has been derived for this 5.8 kbp DNA.

The retroviruses form an integrated genome as a normal and perhaps obligatory step in their replicative cycle. This integrated genome, termed the provirus, is formed by integration of double-stranded (DS) DNA molecules, which are synthesized by reverse transcriptase shortly after infection (Weinberg 1977). Once established, the provirus can serve as template for the synthesis of viral mRNA and progeny virion RNA. The integrated genome also serves to perpetuate the viral presence in the initially infected cell and its progeny. Since most retroviruses are not cytopathic, the infected cell and its progeny replicate normally and the acquired provirus is transmitted as a stable genetic determinant to all descendants of the initially infected cell.

The virion RNAs from Moloney murine leukemia virus (MuLV) and Moloney murine sarcoma virus (MSV) were translated in a micrococcal nuclease-treated cell-free system from rabbit reticulocytes. The predominant polypeptides formed from 35S MuLV RNA were 78,000 and 65,000 daltons in molecular weight, and minor components with molecular weights of 180,000,110,000, 52,000 and 40,000 daltons were also observed. The 30S MSV RNA yielded two predominant polypeptides of 62,000 and 43,000 daltons, and minor components about 72,000, 40,000 and 18,000 daltons in molecular weight. The predominant polypeptides generated by both MuLV and MSV RNA were found to be precursors of the core proteins by immunoprecipitation with specific antisera. The 180,000 dalton molecular weight polypeptide encoded by MuLV RNA was immunoprecipitated both by antisera to the core protein (p30) and reverse transcriptase. The major products therefore appear to be Pr65gag and Pr78gag; an important minor product is Pr180gag-pol. Most of the products of <65,000 daltons synthesized in either system contained sequences from the core protein precursor, but the 43,000 and 18,000 dalton molecular weight polypeptides generated by MSV RNA did not precipitate with antisera to MuLV proteins.

Restriction ondonuclease EcoRI was used to study the structure of the free ribosomal DNA molecules from Tetrahymena pyriformis, strain GL. From the following observations we conclude that the free rDNA molecules from Tetrahymena are giant palindromes‡‡The term palindrome is defined as a word or sentence reading the same in both directions. We apply this term to those double-stranded DNA regions with an axis of 2-fold rotational symmetry, since only such regions will reud (transcribe) identically from bot,h ends of the region, e.g., each containing two genes for preribosomal RNA arranged in rotational symmetry as inverted repeating sequences. Analyses of the sizes of products of partial or complete digestion and quantitative analyses of the products of complete digestion of uniformly 32P-labeled rDNA yielded an RI endonucleolytic cleavage map which showed that the EcoRI recognition sites are arranged symmetrically about the center of the rDNA molecule.