Characterization of a novel human papillomavirus DNA in the cervical carcinoma cell line ME180.

The human cervical carcinoma cell line ME180 was examined for human papillomavirus (HPV) DNA and RNA. The integrated DNA of a presumably new HPV type showing a relationship closer to HPV39 than to HPV18 was cloned and sequenced. HPV sequences from the E6-E7-E1 region are expressed as poly(A)+ RNAs.     

Isolation and analysis of a cDNA clone that encodes an alfalfa (Medicago sativa) aspartate aminotransferase

Summary Nitrate reductase (NR) assays revealed a bi-specific NAD(P)H-NR (EC 1.6.6.2.) to be the only nitrate-reducing enzyme in leaves of hydroponically grown birches. To obtain the primary structure of the NAD(P)H-NR, leaf poly(A)⁺ mRNA was used to construct a cDNA library in the lambda gt11 phage. Recombinant clones were screened with heterologous gene probes encoding NADH-NR from tobacco and squash. A 3.0 kb cDNA was isolated which hybridized to a 3.2 kb mRNA whose level was significantly higher in plants grown on nitrate than in those grown on ammonia. The nucleotide sequence of the cDNA comprises a reading frame encoding a protein of 898 amino acids which reveals 67%–77% identity with NADH-nitrate reductase sequences from higher plants. To identify conserved and variable regions of the multicentre electron-transfer protein a graphical evaluation of identities found in NR sequence alignments was carried out. Thirteen well-conserved sections exceeding a size of 10 amino acids were found in higher plant nitrate reductases. Sequence comparisons with related redox proteins indicate that about half of the conserved NR regions are involved in cofactor binding. The most striking difference in the birch NAD(P)H-NR sequence in comparison to NADH-NR sequences was found at the putative pyridine nucleotide binding site. Southern analysis indicates that the bi-specific NR is encoded by a single copy gene in birch. These sequence data appeared in the EMBL/GenBank/DDBJ nucleotide sequence data bases under the accession number X54097     

COMMENTARY ON ZOHAR'S “PROSPECTS FOR‘GENETIC THERAPY’- CAN A PERSON BENEFIT FROM BEING ALTERED?”

In his paper on the effects of Prenatal Genetic Intervention (PGI) on personal identity, Noam Zohar comes to a conclusion about genetic makeup and the uses of gene therapy quite different from the one I reach in another piece in this issue. Zohar's argument rests on the contention that personal identity changes with alteration of the genome, following what I have identified as the "constitutive" view. To see that this is the pillar supporting the weight of his argument, consider the following. Questions of identity aside, how can it be that altering the genome of children suffering from Lesch-Nyhan syndrome or Tay-Sachs disease so that they now produce the enzyme that they formerly lacked does not benefit them? Clearly, if their identities were not changed, such individuals would in fact realize great benefit from PGI, since the devastating bad effects of the genetic flaw would be avoided. Such a change would certainly make the altered individuals better off, that is, it would benefit them. On this, Zohar and I do not disagree. Persistence of identity through such genetic change is the sticking point.     

Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells

The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of [32P]orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identities of the insulin and IGF I receptor beta-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the beta-subunit of the insulin receptor correlated with occupancy of the beta-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the beta-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the beta-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.     

Expression of aldolase A messenger RNAs in human adult and foetal tissues and in hepatoma

3 specific cDNA clones for human aldolase A were isolated from a human muscle library. One of them was subcloned in M 13 phage, then used as a probe to investigate the patterns and the levels of aldolase A mRNA in various human tissues. Two mRNA species differing in length were observed. The lighter one -1550 bases- was found specific to skeletal muscle; its amount increased during muscle development. The heavier aldolase A mRNA -1650 bases- accounted for foetal and ubiquitous presence of aldolase A isozyme. The resurgence of aldolase A in hepatomas occurred through this latter mRNA species.     

Modifications of the expression of liver-specific and non-specific messenger RNAs during azo-dye hepatocarcinogenesis

The expression of specific and non-specific rat liver messenger RNAs has been studied during 3'-methyl-4-(dimethylamino)azobenzene (3'-MeDAB) carcinogenesis, using cDNA probes complementary to mRNAs encoding aldolase A and B, L-type pyruvate kinase, albumin, alpha-fetoprotein, transferrin and an unidentified 2.7 X 10(3)-base mRNA. mRNAs specific for undifferentiated cells, such as those encoding aldolase A and the unidentified 2.7 X 10(3)-base species were re-expressed very early, being easily detectable at the 1st week of 3'-MeDAB treatment. They reached a maximum of expression at the 4th week. Simultaneously the levels of aldolase B and L-type pyruvate kinase mRNAs dramatically decreased as compared to controls, but remained responsive to induction by a high-carbohydrate diet. Albumin and transferrin mRNA levels were only slightly modified in the course of the carcinogenic diet. At the terminal stage of hepatocarcinogenesis, i.e. in malignant hepatoma cells, expression and inducibility of aldolase B and L-type pyruvate kinase mRNAs were similar to those in normal adult rats while mRNAs specific for undifferentiated or foetal stages were also synthesized. The very early changes in gene expression for aldolases A and B, L-type pyruvate kinase and the 2.7 X 10(3)-base mRNA species could indicate that carcinogenic diet modifies gene control mechanisms long before inducing hepatoma.     

How do retroviral oncogenes induce transformation in avian erythroid cells?

The v-erb B oncogene, as well as other oncogenes of the src-gene family transform immature erythroid cells from chick bone marrow in vivo and in vitro. The erb B-transformed erythroid cells differ from normal late erythroid precursors (CFU-E) in that they have acquired the capacity to undergo self-renewal as well as to differentiate terminally. They also do not require the normal erythroid differentiation hormone, erythropoietin, for either process. Cooperation of v-erb B with a second oncogene, v-erb A, results in a differentiation arrest of the transformed cells, which now only use the self-renewal pathway. Studies with conditional and non-conditional mutants in both v-erb B and v-erb A will be presented to elucidate further how the transforming proteins encoded by these oncogenes, gp74erb B and gp75gag-erb A, affect the differentiation programme of the infected erythroid precursor with the outcome of hormone-independent leukaemic cells arrested at an early stage of erythroid differentiation.     

Gene fusion vehicles for the analysis of gene expression in Rhizobium meliloti.

A set of plasmid cloning vehicles was developed to facilitate the construction of gene or operon fusions in Rhizobium meliloti. The vehicles also contain a broad-host-range replicon and could be introduced into bacteria either by transformation or by transduction, using bacteriophage P2. Insertion of foreign DNA into a unique restriction endonuclease cleavage site promotes the synthesis of either the Escherichia coli lactose operon or the kanamycin phosphotransferase gene from transposon Tn5. Expression of the lactose operon could be detected by observing the color of Rhizobium colonies on medium that contained a chromogenic indicator. We also determined the growth conditions that make it possible to select either for or against the expression of the E. coli lactose operon in R. meliloti. Recombinant plasmids were constructed by inserting MboI restriction fragments of R. meliloti DNA into one of the vehicles, pMK353 . Expression of beta-galactosidase by a number of these recombinants was measured in both R. meliloti and E. coli.