RESUMO
Accounting for energy use by fishes has been taking place for over 200 years. The original, and continuing gold standard for measuring energy use in terrestrial animals, is to account for the waste heat produced by all reactions of metabolism, a process referred to as direct calorimetry. Direct calorimetry is not easy or convenient in terrestrial animals and is extremely difficult in aquatic animals. Thus, the original and most subsequent measurements of metabolic activity in fishes have been measured via indirect calorimetry. Indirect calorimetry takes advantage of the fact that oxygen is consumed and carbon dioxide is produced during the catabolic conversion of foodstuffs or energy reserves to useful ATP energy. As measuring [CO2 ] in water is more challenging than measuring [O2 ], most indirect calorimetric studies on fishes have used the rate of O2 consumption. To relate measurements of O2 consumption back to actual energy usage requires knowledge of the substrate being oxidized. Many contemporary studies of O2 consumption by fishes do not attempt to relate this measurement back to actual energy usage. Thus, the rate of oxygen consumption (MËO2 ) has become a measurement in its own right that is not necessarily synonymous with metabolic rate. Because all extant fishes are obligate aerobes (many fishes engage in substantial net anaerobiosis, but all require oxygen to complete their life cycle), this discrepancy does not appear to be of great concern to the fish biology community, and reports of fish oxygen consumption, without being related to energy, have proliferated. Unfortunately, under some circumstances, these measures can be quite different from one another. A review of the methodological history of the two measurements and a look towards the future are included.
Assuntos
Calorimetria Indireta , Metabolismo Energético , Peixes/fisiologia , Consumo de Oxigênio , Animais , Calorimetria Indireta/história , Dióxido de Carbono/metabolismo , História do Século XVIII , História do Século XIX , História do Século XX , Oxigênio/metabolismoRESUMO
Individual striped bass Morone saxatilis were each exposed in random order to aquatic hypoxia (10% air saturation) either while swimming at 50% of their estimated critical swimming speed (Ucrit ) or while at rest until they lost equilibrium. Individuals were always less tolerant of hypoxia when swimming (P < 0.01); the average fish was over five times more tolerant to the same hypoxia exposure when not swimming. There was no relationship between an individual's rank order of hypoxia tolerance (HT) under the two flow regimes, suggesting that different factors determine an individual's HT when at rest than when swimming.
Assuntos
Bass/fisiologia , Comportamento Animal , Hipóxia , Natação , Animais , Movimentos da ÁguaRESUMO
Several taxonomically disparate groups of fishes have evolved the ability to extract oxygen from the air with elements of their gut. Despite perceived difficulties with balancing digestive and respiratory function, gut air breathing (GAB) has evolved multiple times in fishes and several GAB families are among the most successful fish families in terms of species numbers. When gut segments evolve into an air-breathing organ (ABO), there is generally a specialized region for exchange of gases where the gut wall has diminished, vascularization has increased, capillaries have penetrated into the luminal epithelium and surfactant is produced. This specialized region is generally separated from digestive portions of the gut by sphincters. GAB fishes tend to be facultative air breathers that use air breathing to supplement aquatic respiration in hypoxic waters. Some hindgut breathers may be continuous, but not obligate air breathers (obligate air breathers drown if denied access to air). Gut ABOs are generally used only for oxygen uptake; CO2 elimination seems to occur via the gills and skin in all GAB fishes studied. Aerial ventilation in GAB fishes is driven primarily by oxygen partial pressure of the water (PO2) and possibly also by metabolic demand. The effect of aerial ventilation on branchial ventilation and the cardiovascular system is complex and generalizations across taxa or ABO type are not currently possible. Blood from GAB fishes generally has a low blood oxygen partial pressure that half saturates haemoglobin (p50) with a very low erythrocytic nucleoside triphosphate concentration [NTP]. GAB behaviour in nature depends on the social and ecological context of the animal as well as on physiological factors.
Assuntos
Peixes/fisiologia , Trato Gastrointestinal/fisiologia , Respiração , Ar , Animais , Evolução Biológica , Trato Gastrointestinal/irrigação sanguínea , Brânquias/fisiologia , Hipóxia , Oxigênio/sangue , Consumo de OxigênioRESUMO
A major problem in the study of lesions of atherosclerosis is the difficulty of imaging noninvasively the lesions and following their progression in vivo. To address this problem, we have developed advanced magnetic resonance techniques to noninvasively and serially image advanced lesions of atherosclerosis in the rabbit abdominal aorta. Both lumen and wall were imaged with high resolution. Progression of disease, resulting in increase in lesion mass, decrease in arterial lumen, or stenosis, and intralesion complications, can be detected. Images acquired in vivo correlate with the fine structure of the lesions of atherosclerosis, including the fibrous cap, necrotic core, and lesion fissures, as verified by gross examination, dissection microscopy, and histology. The ability to noninvasively identify the features of atherosclerotic plaques, has significant implications for determining risks and benefits associated with different therapeutic approaches.
Assuntos
Arteriosclerose/diagnóstico , Angiografia por Ressonância Magnética/métodos , Animais , Arteriosclerose/patologia , Dieta Aterogênica , Células Espumosas , Masculino , Coelhos , Fatores de TempoRESUMO
The HIV-1-specific cytotoxic T lymphocyte (CTL) response is temporally associated with the decline in viremia during primary HIV-1 infection, but definitive evidence that it is of importance in virus containment has been lacking. Here we show that in a patient whose early CTL response was focused on a highly immunodominant epitope in gp 160, there was rapid elimination of the transmitted virus strain and selection for a virus population bearing amino acid changes at a single residue within this epitope, which conferred escape from recognition by epitope-specific CTL. The magnitude (> 100-fold), kinetics (30-72 days from onset of symptoms) and genetic pathways of virus escape from CTL pressure were comparable to virus escape from antiretroviral therapy, indicating the biological significance of the CTL response in vivo. One aim of HIV-1 vaccines should thus be to elicit strong CTL responses against multiple codominant viral epitopes.
Assuntos
Síndrome da Imunodeficiência Adquirida/virologia , HIV-1/imunologia , Linfócitos T Citotóxicos/imunologia , Viremia/imunologia , Síndrome da Imunodeficiência Adquirida/imunologia , Proteína gp160 do Envelope de HIV/imunologia , Humanos , Epitopos Imunodominantes/imunologia , Sondas de OligonucleotídeosRESUMO
Human immunodeficiency virus (HIV)-1 infection is associated with the development of aggressive extranodal B-cell non-Hodgkin's lymphomas. Using microvascular endothelial cell (MVEC)-enriched bone marrow stromal cultures, HIV infection of stromal MVECs from lymphoma patients induced the outgrowth of malignant B cells. MVECs were the only HIV-infected cells in the stroma, and purified brain MVECs also induced a phenotype supportive of neoplastic B-cell attachment and proliferation. HIV infection of MVECs stimulated surface expression of CD40 and allowed preferential induction of the vascular cell adhesion molecule VCAM-1 after CD40 triggering. B-lymphoma cells expressed the CD40 ligand (CD40L), and blocking of CD40-CD40L interactions between HIV-infected MVECs and B-lymphoma cells inhibited B-cell attachment and proliferation. These observations suggest that HIV promotes B-lymphoma cell growth through facilitating attachment of lymphoma cells to HIV-infected MVECs and represent a novel mechanism through which viruses may induce malignancies.
Assuntos
Antígenos CD40/biossíntese , Endotélio Vascular/imunologia , HIV-1/imunologia , Linfoma Relacionado a AIDS/imunologia , Antígenos CD40/metabolismo , Ligante de CD40 , Células Cultivadas , Circulação Cerebrovascular , Citometria de Fluxo , Humanos , Linfoma Relacionado a AIDS/patologia , Linfoma Relacionado a AIDS/virologia , Glicoproteínas de Membrana/metabolismo , Microcirculação , Microscopia de Fluorescência , Regulação para Cima , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes life-threatening disease in patients who are immunosuppressed for bone marrow or tissue transplantation or who have AIDS (ref. 1). HCMV establishes lifelong latent infections and, after periodic reactivation from latency, uses a panel of immune evasion proteins to survive and replicate in the face of robust, fully primed host immunity. Monocyte/macrophages are important host cells for HCMV, serving as a latent reservoir and as a means of dissemination throughout the body. Macrophages and other HCMV-permissive cells, such as endothelial and glial cells, can express MHC class II proteins and present antigens to CD4+ T lymphocytes. Here, we show that the HCMV protein US2 causes degradation of two essential proteins in the MHC class II antigen presentation pathway: HLA-DR-alpha and DM-alpha. This was unexpected, as US2 has been shown to cause degradation of MHC class I (refs. 5,6), which has only limited homology with class II proteins. Expression of US2 in cells reduced or abolished their ability to present antigen to CD4+ T lymphocytes. Thus, US2 may allow HCMV-infected macrophages to remain relatively 'invisible' to CD4+ T cells, a property that would be important after virus reactivation.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Citomegalovirus/fisiologia , Antígenos de Histocompatibilidade Classe II/imunologia , Proteínas do Envelope Viral/metabolismo , Adenoviridae/genética , Citomegalovirus/genética , Vetores Genéticos , Glioblastoma , Antígenos HLA-D/imunologia , Antígenos HLA-D/metabolismo , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Testes de Precipitina , Frações Subcelulares , Células Tumorais Cultivadas , Proteínas do Envelope Viral/genética , Latência ViralRESUMO
AbstractGymnotiform swimming is a specialized form of swimming wherein thrust is produced by the ribbonlike motion of an elongate anal fin, while the body is held relatively stiff. This form of swimming has been extensively examined in relation to the biomechanics of thrust production, the kinematics of the anal fin, and neuromuscular control, whereas few studies have examined whole-animal performance parameters of this swimming mode. The goals of this research were to (1) assess the maximum abilities and repeatability of two swimming performance measures, sprinting and prolonged swimming, which would indicate that these performance measures in a gymnotiform swimmer may be a target for selection, similar to body-caudal fin-swimming fish; (2) examine how a gymnotiform swimmer modulates swimming speed; and (3) determine whether modulatory behavior is consistent across different-sized fish and within individuals across time. Sprinting and prolonged swimming were examined in black ghost knifefish (Apteronotus albifrons; N=15), multiple times on the same day, and were measured again 4 wk later. Sprinting ability was measured by chasing a fish down a photocell-lined racetrack and obtaining the fastest speed between any 8-cm span. Prolonged swimming abilities were measured in a constant acceleration test (Ucat) in a Brett-style swim tunnel by measuring the maximum speed the fish could attain against a steadily increasing water velocity. We determined frequency, wavelength, and amplitude of the anal fin sine wave in fish swimming at different speeds during the Ucat trials. We found repeatable measures of sprint speed and Ucat performance over short (day) and medium (4 wk) time periods for both tests. Neither sprint nor Ucat performance was significantly dependent on size, suggesting that the primary driver of performance variation was individual differences in physiology. Most modulation of swimming speed occurred through changes in the frequency of the wave train processing down the anal fin, with only modest changes to the wavelength and minimal changes to amplitude. Finally, we compare our measures of swimming performance in this gymnotiform swimmer to published values of body-caudal fin swimmers to demonstrate that this form of locomotion results in comparable sprint and constant-acceleration values.
Assuntos
Peixes/fisiologia , Natação/fisiologia , Animais , Fenômenos Biomecânicos , HumanosRESUMO
Studies of inter-individual variation in fish swimming performance may provide insight into how selection has influenced diversity in phenotypic traits. We investigated individual variation and short-term repeatability of individual swimming performance by wild European sea bass in a constant acceleration test (CAT). Fish were challenged with four consecutive CATs with 5 min rest between trials. We measured maximum anaerobic speed at exhaustion (U(CAT)), gait transition speed from steady aerobic to unsteady anaerobic swimming (U(gt)), routine metabolic rate (RMR), post-CAT maximum metabolic rate (MMR), aerobic scope and recovery time from the CATs. Fish achieved significantly higher speeds during the first CAT (U(CAT)=170 cm s(-1)), and had much more inter-individual variation in performance (coefficient of variation, CV=18.43%) than in the subsequent three tests (U(CAT)=134 cm s(-1); CV=7.3%), which were very repeatable among individuals. The individual variation in U(CAT) in the first trial could be accounted for almost exclusively by variation in anaerobic burst-and-coast performance beyond U(gt). The U(gt) itself varied substantially between individuals (CV=11.4%), but was significantly repeatable across all four trials. Individual RMR and MMR varied considerably, but the rank order of post-CAT MMR was highly repeatable. Recovery rate from the four CATs was highly variable and correlated positively with the first U(CAT) (longer recovery for higher speeds) but negatively with RMR and aerobic scope (shorter recovery for higher RMR and aerobic scope). This large variation in individual performance coupled with the strong correlations between some of the studied variables may reflect divergent selection favouring alternative strategies for foraging and avoiding predation.
Assuntos
Bass/fisiologia , Metabolismo Energético , Natação/fisiologia , Aceleração , Animais , Consumo de OxigênioRESUMO
Human cytomegalovirus (HCMV) is associated with the acceleration of a number of vascular diseases such as atherosclerosis, restenosis, and transplant vascular sclerosis (TVS). All of these diseases are the result of either mechanical or immune-mediated injury followed by inflammation and subsequent smooth muscle cell (SMC) migration from the vessel media to the intima and proliferation that culminates in vessel narrowing. A number of epidemiological and animal studies have demonstrated that CMV significantly accelerates TVS and chronic rejection (CR) in solid organ allografts. In addition, treatment of human recipients and animals alike with the antiviral drug ganciclovir results in prolonged survival of the allograft, indicating that CMV replication is a requirement for acceleration of disease. However, although virus persists in the allograft throughout the course of disease, the number of directly infected cells does not account for the global effects that the virus has on the acceleration of TVS and CR. Recent investigations of up- and downregulated cellular genes in infected allografts in comparison to native heart has demonstrated that rat CMV (RCMV) upregulates genes involved in wound healing (WH) and angiogenesis (AG). Consistent with this result, we have found that supernatants from HCMV-infected cells (HCMV secretome) induce WH and AG using in vitro models. Taken together, these findings suggest that one mechanism for HCMV acceleration of TVS is mediated through induction of secreted cytokines and growth factors from virus-infected cells that promote WH and AG in the allograft, resulting in the acceleration of TVS. We review here the ability of CMV infection to alter the local environment by producing cellular factors that act in a paracrine fashion to enhance WH and AG processes associated with the development of vascular disease, which accelerates chronic allograft rejection.
Assuntos
Infecções por Citomegalovirus/patologia , Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Doenças Vasculares/virologia , Animais , Citocinas/biossíntese , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Ratos , EscleroseRESUMO
In situ hybridization was used to detect human cytomegalovirus (HCMV) in the peripheral blood mononuclear cells of some naturally infected (seropositive) individuals. A subpopulation of cells hybridized specifically to a portion of the HCMV genome that is heavily transcribed during the immediate-early period of infection. The hybridization signal was markedly reduced by base hydrolysis and ribonuclease, and therefore the probe appears to be detecting viral RNA. A fluorescence-activated cell sorter was used to select lymphocytes bearing the OKT4 and OKT8 markers. Hybridization with the HCMV probe revealed a higher proportion of positive cells in the OKT4 than in the OKT8 subset. This observation specifically identifies lymphocytes as a cell population involved in natural HCMV infection and suggests that lymphocytes may be a reservoir for maintaining infection and may also serve as a vehicle for its spread by blood transfusion.
Assuntos
Infecções por Citomegalovirus/microbiologia , Citomegalovirus/isolamento & purificação , Linfócitos/microbiologia , Antígenos de Diferenciação de Linfócitos T , Antígenos de Superfície/análise , Genes Virais , Humanos , Linfócitos/imunologia , Hibridização de Ácido Nucleico , RNA Viral/análise , Replicação ViralRESUMO
Efforts to solve the epidemiologic puzzle of AIDS in Africa are complicated by the presence of multiple human retroviruses. Simple serologic tests that unambiguously distinguish among infections by these retroviruses are essential. To that end, a partially conserved immunoreactive epitope was identified in the transmembrane glycoproteins of human immunodeficiency viruses (HIV) types 1 and 2. Synthetic peptides derived from these conserved domains were used in sensitive and specific immunoassays that detect antibodies in sera from patients infected with HIV-1 or HIV-2. By making single amino acid substitutions in the HIV-1 peptide, it was possible to demonstrate HIV-1 strain-specific antibody responses to this epitope. Such custom-designed peptides synthesized from this domain are likely to detect newly discovered HIV types, define infection with specific HIV strains, and allow detection of group-common antibodies.
Assuntos
Infecções por Deltaretrovirus/diagnóstico , Leucemia de Células Pilosas/diagnóstico , Sequência de Aminoácidos , Infecções por Deltaretrovirus/imunologia , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática/métodos , Glicoproteínas/genética , Humanos , Leucemia de Células Pilosas/imunologia , Peptídeos , Proteínas do Envelope Viral/genéticaRESUMO
Human cytomegalovirus (HCMV) accelerates transplant vascular sclerosis (TVS), a consequence of angiogenesis (AG) and wound repair (WR). While HCMV can be localized to TVS lesions, the low number of infected cells suggests a global effect on target tissues. We used microarray analysis followed by real-time-polymerase chain reaction (RT-PCR) in an RCMV-accelerated TVS rat cardiac transplant model to determine whether CMV activates host WR and AG factors. Dysregulated cellular genes in allografts from RCMV-infected recipients were compared to those from uninfected recipients and native hearts. We demonstrated that RCMV upregulates the genes involved in WR and AG, which was highest during the critical time of TVS acceleration (21-28 days). Using a standard in vitro AG assay, virus and serum-free supernatants collected at 48 h postinfection significantly induced endothelial cell (EC) migration, branching and tubule formation compared to supernatants from mock-infected cells. Supernatants from ultraviolet (UV)-inactivated RCMV-infected cells failed to induce AG, indicating that virus replication is required. Upregulation of WR and AG genes occurs during the critical period of CMV-accelerated TVS. Targeting these genes may prevent this process and improve allograft survival.
Assuntos
Doença da Artéria Coronariana/complicações , Infecções por Citomegalovirus/complicações , Transplante de Coração/fisiologia , Neovascularização Fisiológica , Cicatrização , Animais , Doença da Artéria Coronariana/virologia , Citomegalovirus , Modelos Animais de Doenças , Genoma , Masculino , Metaloproteinases da Matriz/genética , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante HomólogoRESUMO
Monocytes/macrophages are key cells in the pathogenesis of human cytomegalovirus (HCMV). Although HCMV infection in monocytes is restricted to early events of gene expression, productive infection has been demonstrated in differentiated macrophages in vitro. We examined the cellular and cytokine components that are essential for HCMV replication in Concanavalin A-stimulated monocyte-derived macrophages (MDM). By negative selection, depletion of CD8+ T lymphocytes, but not CD4+ T lymphocytes, CD19+ B cells, or CD56+ NK cells, resulted in a 60-70% reduction in the number of HCMV-infected MDM, and a 4 log decrease in virus production. Neutralization of IFN-gamma and TNF-alpha, but not IL-1, IL-2, or TGF-beta, decreased production of virus by 4 logs and 2 logs, respectively. Subsequently, addition of recombinant IFN-gamma or TNF-alpha to purified monocyte cultures was sufficient to produce HCMV-permissive MDM. While IFN-gamma and TNF-alpha possess antiviral properties, addition of these cytokines to permissive MDM cultures did not affect production of HCMV. Thus, rather than inhibiting replication of HCMV, IFN-gamma and TNF-alpha specifically induce differentiation of monocytes into HCMV-permissive MDM, which are resistant to the antiviral effects of these cytokines.
Assuntos
Antivirais/farmacologia , Citomegalovirus/fisiologia , Interferon gama/farmacologia , Macrófagos/virologia , Fator de Necrose Tumoral alfa/farmacologia , Replicação Viral , Linfócitos T CD8-Positivos/imunologia , Comunicação Celular , Diferenciação Celular , Concanavalina A/farmacologia , Citomegalovirus/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Interferon gama/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Mitógenos/farmacologia , Monócitos/citologia , Fator de Necrose Tumoral alfa/imunologia , Replicação Viral/efeitos dos fármacosRESUMO
Deficiency of erythrocytic and lymphocytic adenosine deaminase (ADA) occurs in some patients with severe combined immunodeficiency disease (SCID). SCID with ADA deficiency is inherited as an autosomal recessive trait. ADA is markedly reduced or undetectable in affected patients (homozygotes), and approximately one-half normal levels are found in individuals heterozygous for ADA deficiency. The metabolism of purine nucleosides was studied in erythrocytes from normal individuals, four ADA-deficiency patients, and two heterozygous individuals. ADA deficiency in intake erythrocytes was confirmed by a very sensitive ammonia-liberation technique. Erythrocytic ADA activity in three heterozygous individuals (0.07,0.08, and 0.14 mumolar units/ml of packed cells) was between that of the four normal controls (0.20-0.37 mumol/ml) and the ADA-deficient patients (no activity). In vitro, adenosine was incorporated principally into IMP in the heterozygous and normal individuals but into the adenosine nucleotides in the ADa-deficient patients. Coformycin (3-beta-D-ribofuranosyl-6,7,8-trihydroimidazo[4,5-4] [1,3] diazepin-8 (R)-ol), a potent inhibitor of ADA, made possible incorporation of adenosine nucleotides in the ADA-deficient patients...
Assuntos
Adenosina Desaminase/deficiência , Eritrócitos/metabolismo , Síndromes de Imunodeficiência/sangue , Nucleosídeo Desaminases/deficiência , Nucleosídeos de Purina/metabolismo , Adenosina Desaminase/sangue , Inibidores de Adenosina Desaminase , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Azepinas/farmacologia , Pré-Escolar , Eritrócitos/enzimologia , Feminino , Formicinas/metabolismo , Glicólise , Guanosina/metabolismo , Humanos , Lactente , Inosina Monofosfato/metabolismo , Nucleotídeos de Inosina/metabolismo , Masculino , Nucleosídeos de Purina/antagonistas & inibidores , Ribonucleosídeos/farmacologia , TioguaninaRESUMO
Human teratocarcinoma cells were used to examine structural features associated with expression of the major immediate-early (IE) gene of human cytomegalovirus. By immunofluorescence, comparison of RNA levels, and in vitro transcription of nuclei, we showed that the major IE gene is inactive in undifferentiated but active in differentiated cells. Therefore, the block in human cytomegalovirus replication in teratocarcinoma cells appears to be at the transcriptional level, in one of the initial genes transcribed. In addition, the in vitro transcription experiments demonstrated that in permissive infections the gene was transcriptionally inactive late in infection. A comparison of the structural features of the promoter region with the active and inactive IE genes showed the presence of constitutive and inducible DNase I-hypersensitive sites. The majority of the constitutive sites existed at -175, -275, -375, -425, and -525 relative to the cap site in an area which has been shown to be capable of simian virus 40 enhancer function. In contrast, the inducible DNase I sites were located outside this region at -650, -775, -875, and -975.
Assuntos
Antígenos de Superfície/genética , Antígenos Virais/genética , Citomegalovirus/genética , Genes Virais , Genes , Proteínas Imediatamente Precoces , Transcrição Gênica , Linhagem Celular , Desoxirribonuclease I/metabolismo , Humanos , Hibridização de Ácido Nucleico , Regiões Promotoras Genéticas , Teratoma , TransfecçãoRESUMO
We have used homologous recombination to disrupt the nuclear gene NIT8 in Chlamydomonas reinhardtii. This is the first report of targeted gene disruption of an endogenous locus in C. reinhardtii and only the second for a photosynthetic eukaryote. NIT8 encodes a protein necessary for nitrate and nitrite assimilation by C. reinhardtii. A disruption vector was constructed by placing the CRY1-1 selectable marker gene, which confers emetine resistance, within the NIT8 coding region. nit8 mutants are unable to grow on nitrate as their sole nitrogen source (Nit-) and are resistant to killing by chlorate. One of 2,000 transformants obtained after selection on emetine-chlorate medium contained a homologous insertion of five copies of the disruption plasmid into the NIT8 gene, producing an emetine-resistant, chlorate-resistant Nit- phenotype. The mutant phenotype was rescued by the wild-type NIT8 gene upon transformation. Seven other mutations at the nit8 locus, presumably resulting from homologous recombination with the disruption plasmid, were identified but were shown to be accompanied by deletions of the surrounding genomic region.
Assuntos
Proteínas de Transporte/genética , Chlamydomonas reinhardtii/genética , Marcação de Genes/métodos , Mutagênese , Proteínas Nucleares/genética , Proteínas de Protozoários , Animais , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Cloratos/farmacologia , Emetina/farmacologia , Genes de Protozoários/genética , Marcadores Genéticos , Vetores Genéticos/genética , Peptídeos e Proteínas de Sinalização Intracelular , Família Multigênica/genética , Nitratos , Proteínas Ribossômicas/genética , Transformação GenéticaRESUMO
To analyze the significance of inducible DNase I-hypersensitive sites occurring in the 5'-flanking sequence of the major immediate-early gene of human cytomegalovirus (HCMV), various deleted portions of the HCMV immediate-early promoter regulatory region were attached to the chloramphenicol acetyltransferase (CAT) gene and assayed for activity in transiently transfected undifferentiated and differentiated human teratocarcinoma cells, Tera-2. Assays of progressive deletions in the promoter regulatory region indicated that removal of a 395-base-pair portion of this element (nucleotides -750 to -1145) containing two inducible DNase I sites which correlate with gene expression resulted in a 7.5-fold increase in CAT activity in undifferentiated cells. However, in permissive differentiated Tera-2, human foreskin fibroblast, and HeLa cells, removal of this regulatory region resulted in decreased activity. In addition, attachment of this HCMV upstream element to a homologous or heterologous promoter increased activity three- to fivefold in permissive cells. Therefore, a cis regulatory element exists 5' to the enhancer of the major immediate-early gene of HCMV. This element negative modulates expression in nonpermissive cells but positively influences expression in permissive cells.
Assuntos
Citomegalovirus/genética , Genes Reguladores , Genes Virais , Linhagem Celular , Deleção Cromossômica , Clonagem Molecular , Enzimas de Restrição do DNA , HumanosRESUMO
We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.