Akt1/PKBalpha is required for normal growth but dispensable for maintenance of glucose homeostasis in mice.

The serine-threonine kinase Akt, also known as protein kinase B (PKB), is an important effector for phosphatidylinositol 3-kinase signaling initiated by numerous growth factors and hormones. Akt2/PKBbeta, one of three known mammalian isoforms of Akt/PKB, has been demonstrated recently to be required for at least some of the metabolic actions of insulin (Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., Chu, Q., Crenshaw, E. B., Kaestner, K. H., Bartolomei, M. S., Shulman, G. I., and Birnbaum, M. J. (2001) Science 292, 1728-1731). Here we show that mice deficient in another closely related isoform of the kinase, Akt1/PKBalpha, display a conspicuous impairment in organismal growth. Akt1(-/-) mice demonstrated defects in both fetal and postnatal growth, and these persisted into adulthood. However, in striking contrast to Akt2/PKBbeta null mice, Akt1/PKBalpha-deficient mice are normal with regard to glucose tolerance and insulin-stimulated disposal of blood glucose. Thus, the characterization of the Akt1 knockout mice and its comparison to the previously reported Akt2 deficiency phenotype reveals the non-redundant functions of Akt1 and Akt2 genes with respect to organismal growth and insulin-regulated glucose metabolism.

Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta).

Glucose homeostasis depends on insulin responsiveness in target tissues, most importantly, muscle and liver. The critical initial steps in insulin action include phosphorylation of scaffolding proteins and activation of phosphatidylinositol 3-kinase. These early events lead to activation of the serine-threonine protein kinase Akt, also known as protein kinase B. We show that mice deficient in Akt2 are impaired in the ability of insulin to lower blood glucose because of defects in the action of the hormone on liver and skeletal muscle. These data establish Akt2 as an essential gene in the maintenance of normal glucose homeostasis.

Ribonuclease protection.

The ribonuclease protection assay (RPA) is a sensitive technique for the analysis of total cellular RNA. It involves generating a specific antisense riboprobe, hybridizing the probe to total RNA, removing unprotected RNA by RNases, and finally isolating and analyzing the protected RNA on a denaturing gel. Although the RPA is somewhat more labor-intensive than Northern analysis, it has the advantage of being more sensitive (as little as 0.1 pg of target RNA can be detected with ideal hybridization conditions). RPAs are also more tolerant of partially degraded RNA (provided the area that is protected is intact). Although RPAs are not as sensitive as polymerase chain reaction (PCR)-based RNA analyses, the target RNA is analyzed directly; a reverse transcription step is not required. Finally, the RPA is quantitative as long as the probe is in excess. More important for the study of imprinted genes, the RPA can be designed to detect allele-specific expression of the target gene of interest.

Molecular biology. Mothers setting boundaries.

Certain genes are only expressed at one allele, a phenomenon called imprinting. Although it is well established that one allele of certain imprinted genes is silenced through methylation, this does not appear to be the case for all imprinted genes. In a thoughtful Perspective, Thorvaldsen and Bartolomei discuss new findings showing that insertion of insulator elements (boundary regions) between the promoter of a gene and its enhancer (a sequence that boosts gene expression) may be another way in which genes are silenced during imprinting.

Changes in condom use after HIV diagnosis.

We investigated changes in condom use after HIV diagnosis. The study population comprised 78 asymptomatic HIV-infected subjects recruited from a clinic in Oslo, Norway, during 1988-92. In total, 240 follow-up visits were carried out. The response variable was repeated measurements of whether or not condoms were always used during anal/vaginal intercourse at follow-up. The explanatory variables were: time from HIV diagnosis to follow-up, exposure group (heterosexually infected, homosexual men, or infected through injecting drug use), time of HIV diagnosis (before 1987 or later), and history of condom use before HIV diagnosis. Random effects logistic regression analysis was used to study incremental changes in condom use, accommodating an unbalanced repeated measurement design. The use of condoms increased monotonously over time after HIV diagnosis in all exposure groups. Condom use was least likely among injecting drug users, subjects diagnosed before 1987 and subjects without a history of frequent condom use before HIV diagnosis.

Role of a 461-bp G-rich repetitive element in H19 transgene imprinting.

The molecular mechanism leading to the imprinted expression of genes is poorly understood. While no conserved cis-acting elements have been identified within the known loci, many imprinted genes are located near directly repetitive sequence elements, suggesting that such repeats might play a role in imprinted gene expression. The maternally expressed mouse H19 gene is located approximately 1.5 kb downstream from a 461-bp G-rich repetitive element. We have used a transgenic model to investigate whether this element is essential for H19 imprinting. Previous results demonstrated that a transgene, which contains 14 kb of H19 sequence, exhibits parent-of-origin specific expression and methylation analogous to the endogenous H19 imprinting pattern. Here, we have generated transgenes lacking the G-rich repeat. One transgene, containing a deletion of the G-rich repetitive element but which includes an additional 1.7 kb of 5' H19 sequence, is imprinted similarly to the endogenous H19 gene. To determine whether the G-rich repeat is conserved in other imprinted mammalian H19 homologues, additional 5' flanking sequences were cloned from the rat and human. This element is conserved in the rat but not in human DNA. These results suggest that the 461-bp G-rich repetitive element is not essential for H19 imprinting.

Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2.

Differentially methylated sequences associated with imprinted genes are proposed to control genomic imprinting. A 2-kb region located 5' to the imprinted mouse H19 gene is hypermethylated on the inactive paternal allele throughout development. To determine whether this differentially methylated domain (DMD) is required for imprinted expression at the endogenous locus, we have generated mice harboring a 1.6-kb targeted deletion of the DMD and assayed for allelic expression of H19 and the linked, oppositely imprinted Igf2 gene. H19 is activated and Igf2 expression is reduced when the DMD deletion is paternally inherited; conversely, upon maternal transmission of the mutation, H19 expression is reduced and Igf2 is activated. Consistent with the DMD's hypothesized role of setting up the methylation imprint, the mutation also perturbs allele-specific methylation of the remaining H19 sequences. In conclusion, these experiments show that the H19 hypermethylated 5' flanking sequences are required to silence paternally derived H19. Additionally, these experiments demonstrate a novel role for the DMD on the maternal chromosome where it is required for the maximal expression of H19 and the silencing of Igf2. Thus, the H19 differentially methylated sequences are required for both H19 and Igf2 imprinting.

Prevalence and trends in homosexual behaviour in Norway.

The three main objectives of this study were to estimate the proportion of the Norwegian population with experience of homosexual behaviour, to study the degree of change in sexual practices among homosexual men during a 5-year period and to study the degree of change in sexual practices reported by HIV positive homosexual men from before to after awareness of HIV-positivity. The data comes from two questionnaire surveys (in 1987 and 1992) of sexual behaviour in the general population of Norway and a questionnaire study of sexual behaviour before and after awareness of HIV-positivity among HIV-positive homosexual men taking part in a cohort study starting in 1988 (the Oslo HIV cohort study). Estimation of the proportion of subjects with homosexual experience was made as well as a trend analysis of the number of male sexual partners per year, number of intercourses per month, condom use and anal sex. Among men aged 18 to 60 from the general population, 3.8% reported homosexual practice during lifetime and 1.2% during the past 3 years. Among women, the same percentages were 3.1 and 1.0. In the surveys, the number of male partners per year decreased significantly for men with current homosexual experience from a yearly median of 1.0 in 1987 to 0.3 in 1992 (p = 0.02). Among HIV-positives, the number of male partners decreased from a yearly median of 4.3 before to 1.6 after awareness of HIV-seropositivity (p < 0.01). Among HIV-positives, a significant increase in the use of condoms, a decrease in the number of intercourses and a decrease in the frequency of anal sex was found. The results show that some changes in sexual practice may have occurred among homosexual men in general in the period from 1987 to 1992, and that more significant changes may have occurred for HIV-positive men. The present data do not support other findings of a relapse to more unsafe sex, but suggest that there is still a need to keep modifying behaviour in order to stop the spread of HIV among men who have sex with other men.

Identification of the Zn(II) site in the copper-responsive yeast transcription factor, AMT1: a conserved Zn module.

The N-terminal metal-binding domains of the copper-activated yeast transcription factors, ACE1 and AMT1, bind to specific DNA sequences in a Cu-dependent fashion. Recombinant AMT1 and ACE1 metal-binding domains are isolated as Cu4Zn1-protein complexes. Site-directed mutagenesis of AMT1 was used in this study to map the ligands of the Cu(I) and Zn(II) ions. The results are consistent with the N-terminal halves of AMT1 and ACE1 consisting of two independent submodules, one binding a single Zn(II) ion and the second binding the tetracopper cluster. The basis of this conclusion is, first, that mutations of two cysteinyl codons and a histidyl codon in the first 42 residues of AMT1 do not alter DNA binding. In contrast, serine substitutions at four cysteine positions at codons 43, 61, 90, and 98 abolish DNA binding. We demonstrated previously that population of the Zn(II) site in AMT1 does not alter the ability of the protein to bind DNA but bound Cu(I) ions are essential for DNA binding [Thorvaldsen, J. L., et al. (1994) Biochemistry 33, 9566-9577]. Second, mutations in the N-terminal 42 residue segment reduce the Zn(II) content of purified mutant AMT1 molecules. Third, a synthetic peptide consisting of the N-terminal 42 residues in AMT1 forms a stable Zn(II) complex and substitution with Co(II) reveals an electronic spectrum identical to that of the Co-substituted intact Cu4AMT1 protein. 113Cd(II) NMR studies reveal that the divalent metal site consists of ligands provided by three cysteinyl thiolates and a single histydyl imidazole. The sequence homology between AMT1, ACE1, and MAC1 in the N-terminal 42 residues suggests that ACE1 and MAC1 will, likewise, contain N-terminal Zn modules. A 42-residue ACE1 synthetic peptide gives identical metal binding properties to the corresponding AMT1 synthetic peptide. Thus, AMT1 and likely ACE1 consist of two contiguous modules, residues 1-42 forming an independent Zn(II) module and residues 43-110 enfolding a tetracopper cluster.

Analysis of copper-induced metallothionein expression using autonomously replicating plasmids in Candida glabrata.

Candida glabrata strains and a stable plasmid were developed that were suitable for analysis of copper-inducible expression from promoters of the three metallothionein (MT) genes. The two homologous MTII genes, MTIIa and MTIIb, encode the same polypeptide but are differentially induced by copper salts. MTIIb is more highly inducible than MTIIa and cells harboring a single MTIIb exhibit a greater resistance to copper salts compared to cells harboring a single MTIIa. The differential copper inducibility was mapped to sequences between -03 and -292 upstream of the MT coding sequences. Expression of MTI is highly Cu-regulated, but this MT gene confers much less resistance than MTII genes.

[Steady homosexual relationship and HIV infection]. / Fast homoseksuelt forhold og HIV-smitte.

In order to study the relative impact of a steady homosexual relationship on the presence of HIV antibodies, a cross-sectional study was undertaken of 719 homosexual men in Oslo during the period of 1983-1987. 74/719 (10%) were HIV positive. 37/74 (50%) of the HIV positives and 185/645 (29%) of the HIV negatives were having a steady homosexual relationship that had lasted six months or more (chi-square test, p < 0.05). In a multivariate logistic regression analysis the adjusted odds ratio of being HIV positive was 2.3 (1.3-4.3, 95% confidence interval (CI)) for homosexual relationship lasting 1/2-4 years and 3.1 (1.5-6.6, 95% CI) for homosexual relationship lasting 5 years or more as compared with not being in a homosexual relationship. The number of male partners and frequent anal intercourse were also significantly associated with presence of HIV antibodies. The results suggest that situational variables associated with a steady partnership may be barriers to HIV-preventive measures.

Mixed Cu+ and Zn2+ coordination in the DNA-binding domain of the AMT1 transcription factor from Candida glabrata.

AMT1 is the transcription factor required for Cu-induced expression of metallothionein genes in the yeast Candida glabrata. The copper-binding, DNA-binding domain of AMT1 has been purified after expression of an AMT1 synthetic gene in bacteria and was confirmed as active in a gel shift assay. The Cu-activated AMT1 was shown to contain a Cu(+)-thiolate tetracopper center and a single Zn2+ site. AMT1 is purified as a Cu-Zn protein from bacterial cultures grown in the presence of CuSO4. Chemical analysis suggested that 4.2 +/- 0.2 and 1.2 +/- 0.2 molar equiv copper and zinc ions bound, respectively. Electrospray mass spectrometry was used to verify that a uniform species was present with 4 Cu+ ions and 1 Zn2+ ion bound per AMT1 molecule. Cu+ binding to form a tetracopper center occurs cooperatively as shown by electrospray MS of apoAMT1 samples reconstituted with increasing equivalency of Cu+. Copper-thiolate coordination was indicated by Cu-S charge-transfer transitions in the ultraviolet, luminescence typical of Cu-thiolate clusters and EXAFS. Analysis of the EXAFS of CuZnAMT1 revealed predominantly trigonal Cu+ coordination and the presence of a polycopper cluster by virtue of a short Cu-Cu distance of 2.7 A. Zn K-edge EXAFS of Cu4Zn1AMT1 and electronic spectroscopy of AMT1 with Co2+ substituted for the single Zn2+ ion are consistent with tetrahedral Zn2+ coordination with thiolate ligands. The Cu-activated AMT1 exhibited a conformation distinct from that of metal-free AMT1 as shown by circular dichroism. DNA binding by AMT1 was dependent on the tetracopper center but was independent of occupancy of the Zn2+ site. This is the first report of a single, uniform tetracopper center in a metal-activated transcription factor.

Heterologous gene expression and protein secretion from Candida glabrata.

We have examined heterologous protein secretion from Candida glabrata with the aid of a stable C. glabrata vector and a secretion reporter cassette comprising the Saccharomyces cerevisiae PGK-gene promoter and a Kluveromyces Iactis secretion signal to drive secretion of Escherichia coli beta-lactamase. Abundant secretion of beta-lactamase from C. glabrata indicates that the S. cerevisiae PGK promoter functions in C. glabrata. Furthermore, we show that C. glabrata processes the secreted beta-lactamase in a manner similar to, but not identical with, S. cerevisiae and K. lactis. C. glabrata may be a suitable new host for the expression of foreign genes.

[Hepatitis C virus infections among persons attending special clinics dealing with problems related to social conditions]. / Hepatitt C-virusinfeksjoner blant personer som i 1990 oppsøkte Miljøetatens legekontorer i Oslo.

We have studied the prevalence of hepatitis C virus antibodies among 526 persons who attended an HIV diagnostic and counselling clinic and a clinic for sexually transmitted diseases in Oslo, Norway, during a 4-month period in early 1990. Possible risk factors for contracting hepatitis C virus infection were analysed and compared with results of the anti-hepatitis C virus test. The over-all prevalence rate of positive anti-hepatitis C virus tests was 7%. The prevalence rate was highest, 70%, among intravenous drug users (26/37). When intravenous drug users were excluded, only 2% of the remaining 484 persons had antibodies to hepatitis C virus. Among persons with no history of intravenous drug use, a positive correlation was found among women between the presence of hepatitis C virus antibodies and previous gonorrhoea. Male homosexual activity did not correlate with the presence of hepatitis C virus infection. Neither the number of heterosexual partners, nor sex with intravenous drug users, correlated with the presence of hepatitis C virus antibodies. Thus, according to our study the overwhelming risk factor for contracting hepatitis C virus infection was intravenous drug use.

Regulation of metallothionein genes by the ACE1 and AMT1 transcription factors.

The AMT1 metalloregulatory trans-acting factor from Candida glabrata was found to functionally mimic the ACE1 metalloregulatory trans-acting factor from Saccharomyces cerevisiae in the copper-induced expression of the chromosomal S. cerevisiae metallothionein gene. Plasmid constructs with promoters of various metal-inducible genes fused to the bacterial beta-galactosidase (lacZ) reporter gene were used in S. cerevisiae to evaluate the roles of ACE1 and AMT1 in mediating metal-stimulated expression. Promoters from the S. cerevisiae CUP1 gene and Cu,Zn-superoxide dismutase (SOD1) and from the C. glabrata MT genes MTI, MTIIa, and MTIIb were used. The ACE1 factor was effective in the metalloregulation of the two S. cerevisiae promoters, CUP1 and SOD1, but of only one C. glabrata promoter, MTI. AMT1 was found to be effective in the metalloregulation of all three C. glabrata MT promoters and the two S. cerevisiae promoters tested. The regulation mediated by both ACE1 and AMT1 was copper-dependent and copper-specific. Episomally expressed SWI5, a distinct trans-acting factor of S. cerevisiae, enhanced only the basal expression from promoters. The SWI5 enhancement was not metal dependent. In conclusion, AMT1 and ACE1 are functionally homologous in metal-specific regulation, AMT1 appears to be more promiscuous than ACE1 in this function.

Disruption analysis of metallothionein-encoding genes in Candida glabrata.

Candida glabrata harbors multiple genes encoding metallothionein (MT). We have disrupted MT-IIa, an amplified locus, and MT-IIb, a single-copy gene, to determine the roles of various MT genes in CuSO4 resistance in C. glabrata. The concentration of CuSO4 required to inhibit the growth by 50% (IC50) of a C. glabrata strain harboring an amplified MT-IIa locus and a single-copy MT-IIb and MT-I genes was 7 mM in a synthetic complete medium. The IC50 decreased to approx. 1 mM when the amplified MT-IIa locus was deleted. The disruption of the MT-IIb gene decreased the IC50 further to 0.1 mM. The CuSO4 resistance in a strain lacking both of the MT-II genes was attributable to MT-I; no evidence was found for the production of (gamma EC)nG isopeptides. The comparison of the nucleotide sequence of MT-IIb to that of MT-IIa revealed the same coding sequence with differences in the 5' region. However, substantial differences were found in the 3' region. MT-IIb was expressed since we were able to purify the protein from the strain that had an intact MT-IIb gene, but a deleted MT-IIa gene. In addition, CuSO4 resistance was provided by MT-IIb. Northern analysis of the total RNA from varied C. glabrata strains indicated no significant changes in the expression of MT-I in the presence or absence of the MT-II genes.

Cloning system for Candida glabrata using elements from the metallothionein-IIa-encoding gene that confer autonomous replication.

The yeast Candida glabrata harbors two distinct gene families that encode metallothioneins (MTs). One of these loci, the MT-IIa locus, exhibits selective and tandem amplification in many wild type strains of C. glabrata. The present paper demonstrates that the amplified MT-IIa gene contains autonomously replicating sequences (ARS). These ARS elements have been used to construct vectors capable of replicating in C. glabrata. The ARS element(s) in the MT-IIa gene were localized to a 457-bp segment downstream from the MT-IIa coding sequence. Although plasmids containing this fragment transform C. glabrata with high frequency, the stability of the transformants and the copy number of the plasmid improve when the entire 1.25-kb MT-IIa gene is used. Transformation of C. glabrata with plasmids carrying the 2 microns circle ARS of Saccharomyces cerevisiae led to the formation of micro-colonies, indicating that the ARS elements of 2 microns plasmids replicate only to a limited extent in C. glabrata. Conversely, a C. glabrata plasmid carrying three copies of the MT-IIa gene was able to transform S. cerevisiae.

Age-related increase in methylation of ribosomal genes and inactivation of chromosome-specific rRNA gene clusters in mouse.

An age-related increase in DNA methylation of the multicopy 18S and 28S ribosomal RNA genes was found in CBA/Ca mice beginning between 6 and 18 months of age at the 5' end of these genes in liver, brain and spleen. The highest level of age-associated hypermethylation was mapped to the proximal 5' spacer domain. Silver staining of actively transcribing ribosomal genes in metaphase chromosomes from stimulated spleen cells provided cytological evidence that these mice have 3 rRNA cistrons located on chromosomes 15, 16, and 18. The ribosomal gene cluster located on chromosome 16 was preferentially inactivated in older animals. Exposure of spleen cells from older individuals to 5-azacytidine appeared to both reactivate ribosomal gene clusters and reduce rRNA gene methylation.