Biomedical subjects

Li Liu

Publications and source records attributed to Li Liu.

At least 343 records · Page 19Linked to original sources

PubMed2002

A conserved family of doublesex-related genes from fishes.

The sex-determining gene Mab-3 of C. elegans and the doublesex gene of Drosophila each contain a common DM domain and share a similar role. Human doublesex-related gene DMRT1 also encodes a conserved DM-related DNA-binding domain. We present here the amplification of a broad range of DM domain sequences from three fish species using degenerate PCR. Our results reveal unexpected complexity of the DM domain gene family in vertebrates.

Amino Acid Sequence↗

PubMed2002

Model for the rotational contribution to quasielastic neutron scattering spectra from supercooled water.

A model is proposed for the first-, second-, and third-order rotational correlation functions that are required for the computation of the rotational intermediate scattering function for the calculation of incoherent quasielastic neutron scattering (QENS) spectra from supercooled water. The model is tested against molecular-dynamics data generated from an extended-simple-point-charge model of water and is found to be satisfactory. The model can be used as a practical method for extracting rotational relaxation parameters from QENS spectra measured at large Q from supercooled bulk water or interfacial water in porous materials.

Journal Article↗

PubMed2002

Regulation of cyclic AMP in rat pulmonary microvascular endothelial cells by rolipram-sensitive cyclic AMP phosphodiesterase (PDE4).

We report here studies on the regulation of the metabolism of adenosine 3',5'-monophosphate (cAMP) in established and primary cultures of rat pulmonary microvascular endothelial cells (RPMVEC). Inhibition by rolipram, a selective inhibitor of cAMP phosphodiesterase (PDE) of the PDE4 gene family, was required to achieve maximal cAMP accumulation induced by direct or receptor-mediated adenylate cyclase activation when measured by [3H]-adenine prelabeling. Rolipram increased cAMP accumulation more effectively than did forskolin, isoproterenol, or adenosine derivatives alone, although extensive synergy was seen with combined agents. High-affinity PDE4 inhibitors, but not low-affinity or non-selective inhibitors, were effective inducers of cAMP accumulation in intact cells. The maximum effects (i.e. intrinsic activities) of these agents in the intact cell did not correlate with their in vitro PDE4 inhibitory affinities. RPMVEC were shown to express almost exclusively the PDE4 gene family isoforms A6 and B3. Guanosine 3',5'-monophosphate hydrolysis, observed in other types of endothelial cells was not found in early or late passage RPMVEC. Reverse transcription-polymerase chain reaction identification of mRNAse supported these conclusions with the exception that PDE2 and PDE4D mRNA isoform transcripts were present. These studies also support the conclusion that the mechanism of rolipram reversal of rat lung ischemia-reperfusion-induced permeability involves PDE4 inhibition in the microvascular endothelial cells of the lung.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2002

Inhibition of protein geranylgeranylation and RhoA/RhoA kinase pathway induces apoptosis in human endothelial cells.

Geranylgeranylation of RhoA small G-protein is essential for its localization to cell membranes and for its biological functions. Many RhoA effects are mediated by its downstream effector RhoA kinase. The role of protein geranylgeranylation and the RhoA pathway in the regulation of endothelial cell survival has not been elucidated. The hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor lovastatin depletes cellular pools of geranylgeranyl pyrophosphate and farnesol pyrophosphate and thereby inhibits both geranylgeranylation and farnesylation. Human umbilical vein endothelial cells (HUVECs) were exposed to lovastatin (3 microm-30 microm) for 48 h, and cell death was quantitatively determined by cytoplasmic histone-associated DNA fragments as well as caspase-3 activity. The assays showed that lovastatin caused a dose-dependent endothelial cell death. The addition of geranylgeraniol, which restores geranylgeranylation, rescued HUVEC from apoptosis. The geranylgeranyltransferase inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, induced apoptosis in HUVEC. Cell death was also induced by a blockade of RhoA function by exoenzyme C3. In addition, treatment of HUVEC with the RhoA kinase inhibitors Y-27632 and HA-1077 caused dose-dependent cell death. Y-27632 did not inhibit other well known survival pathways, such as NF-kappa B, ERK, and phosphatidylinositol 3-kinase/Akt. However, there was an increase in p53 protein level concomitant with Y-27632-induced cell death. Unlike the apoptosis induced by TNF-alpha, which occurs only with inhibition of new protein synthesis, apoptosis induced by inhibitors of HMG-CoA reductase, geranylgeranyltransferase, or RhoA kinase was blocked by cycloheximide. Our data indicate that inhibition of protein geranylgeranylation and RhoA pathways induce apoptosis in HUVEC and that induction of p53 or other proapoptotic proteins is required for this process.

Apoptosis↗

PubMed2002

Charcot-Leyden crystal protein (galectin-10) is not a dual function galectin with lysophospholipase activity but binds a lysophospholipase inhibitor in a novel structural fashion.

Charcot-Leyden crystal (CLC) protein, initially reported to possess weak lysophospholipase activity, is still considered to be the eosinophil's lysophospholipase, but it shows no sequence similarities to any known lysophospholipases. In contrast, CLC protein has moderate sequence similarity, conserved genomic organization, and near structural identity to members of the galectin superfamily, and it has been designated galectin-10. To definitively determine whether or not CLC protein is a lysophospholipase, we reassessed its enzymatic activity in peripheral blood eosinophils and an eosinophil myelocyte cell line (AML14.3D10). Antibody affinity chromatography was used to fully deplete CLC protein from eosinophil lysates. The CLC-depleted lysates retained their full lysophospholipase activity, and this activity could be blocked by sulfhydryl group-reactive inhibitors, N-ethylmaleimide and p-chloromercuribenzenesulfonate, previously reported to inhibit the eosinophil enzyme. In contrast, the affinity-purified CLC protein lacked significant lysophospholipase activity. X-ray crystallographic structures of CLC protein in complex with the inhibitors showed that p-chloromercuribenzenesulfonate bound CLC protein via disulfide bonds with Cys(29) and with Cys(57) near the carbohydrate recognition domain (CRD), whereas N-ethylmaleimide bound to the galectin-10 CRD via ring stacking interactions with Trp(72), in a manner highly analogous to mannose binding to this CRD. Antibodies to rat pancreatic lysophospholipase identified a protein in eosinophil and AML14.3D10 cell lysates, comparable in size with human pancreatic lysophospholipase, which co-purifies in small quantities with CLC protein. Ligand blotting of human and murine eosinophil lysates with CLC protein as probe showed that it binds proteins also recognized by antibodies to pancreatic lysophospholipase. Our results definitively show that CLC protein is not one of the eosinophil's lysophospholipases but that it does interact with eosinophil lysophospholipases and known inhibitors of this lipolytic activity.

Animals↗

PubMed2002

The human herpes virus 8-encoded viral FLICE inhibitory protein physically associates with and persistently activates the Ikappa B kinase complex.

The human herpesvirus 8 (HHV8, also called Kaposi's sarcoma-associated herpesvirus) has been linked to Kaposi's sarcoma and primary effusion lymphoma (PEL) in immunocompromised individuals. We demonstrate that PEL cell lines have a constitutively active NF-kappaB pathway, which is associated with persistent phosphorylation of IkappaBalpha. To elucidate the mechanism of NF-kappaB activation in PEL cell lines, we have investigated the role of viral FLICE inhibitory protein (vFLIP) in this process. We report that stable expression of HHV8 vFLIP in a variety of cell lines is associated with persistent NF-kappaB activation caused by constitutive phosphorylation of IkappaBalpha. HHV8 vFLIP gets recruited to a approximately 700-kDa IkappaB kinase (IKK) complex and physically associates with IKKalpha, IKKbeta, NEMO/IKKgamma, and RIP. HHV8 vFLIP is incapable of activating NF-kappaB in cells deficient in NEMO/IKKgamma, thereby suggesting an essential role of an intact IKK complex in this process. Our results suggest that HHV8 vFLIP might contribute to the persistent NF-kappaB activation observed in PEL cells by associating with and stimulating the activity of the cellular IKK complex.

Apoptosis↗

PubMed2002

Importance of the treatment package time in surgery and postoperative radiation therapy for squamous carcinoma of the head and neck.

BACKGROUND: To determine the effect of treatment time-related factors on outcome in patients treated with surgery and postoperative radiation therapy (RT) for locally advanced squamous cell carcinoma of head and neck (SCCHN) METHODS: A retrospective review was performed on 208 consecutive patients treated from 1992 to 1997 with surgery and postoperative RT (> or =55 Gy) for SCCHN. The treatment time factors considered were (1) interval from surgery to the start of RT; (2) RT duration; and (3) the total time from surgery to completion of RT (treatment package time). Treatment package time was dichotomized into short (< or =100 days) vs long (>100 days) categories. Other variables considered were clinical and pathologic staging, margin status, RT dose, and tumor site. Patients were also divided into intermediate- and high-risk groups on the basis of eligibility for RTOG 95-01. Univariate (logrank) and multivariate analyses were performed. RESULTS: Median follow-up for surviving patients was 24 months. Actuarial 2-year locoregional control (LRC) and survival rates were 82% and 71%, respectively. In univariate analysis, factors associated with higher locoregional failure were high-risk group (p =.011), margin status (p =.038), pathologic stage (p =.035), clinical N stage (p =.006), package time (p =.013), and RT treatment time (p =.03). Package time was also a significant predictor of survival in univariate analysis (p =.021). The other two individual time factors, tumor factors, and RT dose were not significant. Both risk status and treatment package time were significant factors in a multivariate model of LRC. CONCLUSIONS: A total treatment package time of <100 days is associated with improved tumor control and survival. Every effort should be made to keep the time from surgery to the completion of postoperative RT to <100 days.

Adult↗

PubMed2002

Fimbria-fornix lesion does not affect APP levels and amyloid deposition in the hippocampus of APP+PS1 double transgenic mice.

The deposition of amyloid beta peptides (Abeta) and cholinergic dysfunction are two characteristic features of Alzheimer's disease. Several studies have suggested that a compromised cholinergic transmission can increase the amount of amyloid precursor protein (APP) in the denervated cortex (or hippocampus); however, whether this will increase Abeta production is unknown. To investigate the relation between cholinergic neurotransmission and APP metabolism, and the possible role of cholinergic dysfunction in the development of amyloid neuropathology, we lesioned the fimbria-fornix pathway in APP+PS1 double transgenic mice, at 5 and 7 months of age. Three months and 11 months postlesion, the mice were sacrificed for biochemical and histopathological analyses. The fimbria-fornix transection resulted in a substantial depletion of cholinergic markers in the hippocampus at both time points. Three months postlesion, hippocampal APP and Abeta levels were not significantly changed. At 11 months postlesion, the fimbria-fornix lesion did not result in an alteration in either the hippocampal Abeta levels or the extent of Abeta deposition, as assessed by amyloid plaque counts and image analysis of Abeta load in the 18-month-old APP+PS1 mice. Our findings indicate that APP metabolism in mice may be dissociated from cholinergic neurotransmission rather than related as previously suggested in other mammalian species.

Acetylcholinesterase↗

PubMed2002

Effects of ovariectomy and estrogen treatment on learning and hippocampal neurotransmitters in mice.

This study examined the effects of long-term estrogen treatment (sc 17 beta-estradiol minipellets) on learning in C57BL/6J female and male mice using a position discrimination task in the T-maze and a win-stay task (1/8 arms baited) in the radial arm maze (RAM). In addition, hippocampal monoamines and ChAT activity were measured at the end of the study and correlated to task performance. Female sham-operated (gonadally intact) and ovariectomized (OVX) mice were treated with estrogen either for 7 or 40 days before the behavioral tests and intact male mice for 7 days before the behavioral tests. In sham-operated mice the 40-day estrogen treatment improved RAM performance and in OVX mice both the 7- and 40-day estrogen treatments improved the performance in both maze tasks. The estrogen treatment also improved RAM performance in males. The hippocampal ChAT, NA, 5-HIAA, and DOPAC levels were decreased in OVX mice. Furthermore, the effects of estrogen treatment on the levels of hippocampal 5-HT and its metabolite 5-HIAA were different in sham-operated than in OVX mice. We could find no correlation between cognitive measures and neurochemical variables. This study gives new information about the effects of estrogen on learning and hippocampal neurotransmitters in mice.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2002

Experimental study of assessment on ventricular activation origin and contraction sequence by Doppler tissue imaging.

To evaluate the possibility and accuracy of Doppler tissue image (DTI) on assessment of normal and abnormal ventricular activation and contraction sequence, 9 open chest canine hearts were analyzed by acceleration mode, M-mode, and spectrum mode DTI. Our results showed that: (1) Acceleration mode DTI could show the origin of activation and conduction sequence on line; (2) M-mode DTI revealed that the activation in mid-interventricular septum was earlier than that in mid-left ventricular posterior wall at sinus activation; (3) Spectrum DTI showed the ventricular endocardium was activated earlier than the ventricular epicardium in all segments at sinus rhythm. The earliest site of activation of the normal ventricular wall was at middle interventricular septum; the latest site was at basal-posterior wall; the contraction sequence was different at the different walls; (4) During abnormal ventricular activation, mid-left ventricular posterior wall was activated earliest in accordance with the pacing sites. Abnormal ventricular activation was slower than sinus activation, and the contraction sequence varied at different sites of ventricular wall. It is concluded that DTI can be used to localize the origin of normal or abnormal myocardial activation and to assess the contraction sequence conveniently, accurately and non-invasively.

Animals↗

PubMed2002

Agroinfection as a rapid method for propagating Cowpea mosaic virus-based constructs.

To increase the efficiency of infections with Cowpea mosaic virus (CPMV)-based constructs, clones suitable for agroinfection were constructed. Full-length copies of RNA-1 and RNA-2 were inserted between the sequence of a Cauliflower mosaic virus (CaMV) 35S promoter and a nos terminator and were introduced into the Agrobacterium tumefaciens plasmid, pBINPLUS. Infiltration of leaves of either Nicotiana benthamiana or cowpea (Vigna unguiculata) with a bacterial suspension containing a mixture of the RNA-1- and RNA-2-based plasmids resulted in the plants developing typical CPMV symptoms. To confirm the utility of this approach for use with CPMV-based vectors, a GFP construct based on RNA-2 was adapted for agroinfection. Infiltration of N. benthamiana leaves with a mixture of Agrobacteria containing this construct and the RNA-1 plasmid resulted in high levels of GFP expression. The results demonstrate that agroinfection is a suitable method for the propagation of CPMV-based derivatives.

Agrobacterium tumefaciens↗

PubMed2002

Specific cGMP binding by the cGMP binding domains of cGMP-binding cGMP specific phosphodiesterase.

The structure of cyclic GMP (cGMP)-binding (cGB), cGMP specific phosphodiesterase (PDE5) comprises several domains. We have used RT-PCR methods to clone the noncatalytic cGB domains of PDE5 from human colon cancer cell RNA and constructed glutathione-S-transferase (GST) fusion proteins to express and study the domains. One fragment showed 94% identity to bovine PDE5 and coded for the high affinity cGB domain of PDE5 (Val(156)-Asp(394), cGB-I). Another cloned fragment showed 92% identity to bovine PDE5 and coded for the phosphorylation site plus both high and low affinity cGB domains of PDE5 (Val(36)-Glu(529), cGB-II). Both fragments expressed as GST-cGB fusion proteins bound cGMP specifically, as determined by competitive [3H]-cGMP ligand binding. We found that cGB-I showed high affinity cGMP binding with K(d)=0.33 microM. cGB-II showed two cGMP binding sites with similar affinities and specificity to the native enzyme. cGB-II was phosphorylated by cGMP-dependent protein kinase (PKG) as reported for bovine PDE5. These data show that recombinant regulatory regions of PDE5 form cGB sites similar to native enzyme sites and confirm proposed domain functions. These results establish that recombinant fusion proteins of PDE5 domains may be used to further characterize the structure of PDE5.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2002

A phase I study of SPI-077 (Stealth liposomal cisplatin) concurrent with radiation therapy for locally advanced head and neck cancer.

BACKGROUND: Liposomal cisplatin preparations have two potential advantages over the free drug when combined with radiation therapy (RT): 1) selective tumor localization, improving the therapeutic ratio, and 2) prolonged half-life, allowing more radiosensitization. We performed a Phase I study of Stealth liposomal cisplatin (SPI-077) concurrent with RT for head and neck squamous cell carcinoma (HNSCC). METHODS: Patients with Stage IVa/b HNSCC were treated with SPI-077, given intravenously twice two weeks apart, concurrent with RT (60-72 Gy in 6-7 weeks). The SPI-077 dose was escalated in standard phase I design. RESULTS: Twenty patients received 38 doses of SPI-077, escalated from 20-200 mg/m2 in six dose levels. Two of these patients received one dose because of reversible Grade 3 liver toxicity or rash. Three patients had a Grade 1, and one had a Grade 2 infusion reaction. Four patients had transiently elevated transaminases: Grade I (n = 1), Grade 2 (n = 1), and Grade 3 (n = 2). Grade 3 neutropenia occurred in one patient. There was no ototoxicity, neurotoxicity, or nephrotoxicity. In-field radiation skin and mucosal toxicities did not appear to be intensified. Ten of 17 patients (59%) finishing treatment achieved initial complete response. CONCLUSIONS: Systemic and in-field radiation toxicities of SPI-077 were minimal. Infusion reactions were minimized with a slower and more dilute initial infusion. Further dose escalation was stopped in the absence of dose-limiting toxicity to address the reformulation of the liposomally bound cisplatin. Nonetheless, this study shows that high doses of SPI-077 can be given safely. The potentially beneficial therapeutic ratio suggests that liposomal radiosensitizer preparations warrant further investigation.

Adult↗

PubMed2002

Disrupting effects of polychlorinated biphenyls on gonadal development and reproductive functions in chickens.

Polychlorinated biphenyls (PCBs) are worldwide persistent pollutants that have produced detrimental effects on endocrine function and reproduction in a variety of species. The present study revealed effects of PCBs on gonadal development and functions in chickens of different ages. Aroclor 1254 (0-100 microg/egg) was injected into Hyline chicken eggs before incubation. The adult chickens received Aroclor 1254 by gavage (50 mg/kg BW). It was observed that in day 5 embryos, PCBs resulted in a dose-dependent decrease of primordial germ cell (PGC) numbers, and caused PGCs pyknosis and vacuolation. Clomiphen failed to block the effects of PCBs. In the newly hatched chicken, PCBs induced a marked decrease in area of the transverse sections, diameter and relative area of the testicular tubules. The differentiation of germ cells was retarded after PCB treatment. In contrast, the area of the left ovarian transverse sections, the thickness of ovarian cortex and the number of oocytes increased dramatically in the female chickens after PCB exposure. In the adult chickens, PCBs caused no significant changes in body weight, respiration, heart rate, body temperature, red and white blood cell number, but induced a marked decrease in the testicular weight, and severe damage of the seminiferous tubules. The number of the spermatogenic cells and serum testosterone level were decreased significantly by PCBs. On the contrary, in the laying hens there was no significant effect of PCB on egg quality except a slight decrease in egg weight. These results indicated that PCBs exerted its disrupting effects on chicken reproduction with a sex and stage-related pattern, and in vivo disruption of gonadal development represents a possible model for risk assessment of environmental endocrine disrupters by in ovo treatment.

Administration, Oral↗

PubMed2002

Nasopharyngeal carcinoma treated with external radiotherapy, brachytherapy, and concurrent/adjuvant chemotherapy.

The standard treatment for advanced nasopharyngeal carcinoma (NPC) has become external beam radiation therapy (EBXRT) 70 Gy/7 weeks + 3 cycles of concurrent cisplatin followed by 2 to 3 cycles of adjuvant cisplatin/5-fluorouracil (5-FU). Some reports suggest that the addition of low-dose rate brachytherapy to EBXRT also improves local control. To our knowledge, this is the first report of the "triple" combination of EBXRT, brachytherapy, and concurrent/adjuvant chemotherapy. Eleven patients treated from 1992 to 1998 were evaluated. All patients had stage III/IV (excluding T4 lesions) NPC. Treatment consisted of EBXRT (64-70 Gy/7 weeks), followed by a brachytherapy boost (6-15 Gy delivered 0.5 cm deep to the mucosa). Chemotherapy consisted of concurrent cisplatin (100 mg/m2) and post-XRT adjuvant cisplatin (80 mg/m2) and 5-FU (1,000 mg/m2/day x 4 days) for 2 cycles. All 11 patients were evaluable. The average age was 44 years, and median follow-up was 38 months (range: 23-82 months). Median EBXRT dose was 66 Gy, and median brachytherapy dose was 9 Gy (median total dose: 75 Gy). All patients obtained primary tumor complete response (CR). Two patients required post-XRT neck dissection to achieve regional CR. To date, 10 patients are alive with no evidence of disease. The 3-year actuarial survival is 100%. One patient died at 82 months of a late distant recurrence (at 37 months post-XRT). No patient has had a local or neck failure. Chemoradiation plus brachytherapy offers encouraging survival and local-regional control. Further study of this regimen as an alternative or adjunct to intensity-modulated EBXRT is warranted.

Adolescent↗

PubMed2002

Domain-level differences in microsatellite distribution and content result from different relative rates of insertion and deletion mutations.

Microsatellites (short tandem polynucleotide repeats) are found throughout eukaryotic genomes at frequencies many orders of magnitude higher than the frequencies predicted to occur by chance. Most of these microsatellites appear to have evolved in a generally neutral manner. In contrast, microsatellites are generally absent from bacterial genomes except in locations where they provide adaptive functional variability, and these appear to have evolved under selection. We demonstrate a mutational bias towards deletion (repeat contraction) in a native chromosomal microsatellite of the bacterium Mycoplasma gallisepticum, through the collection and analysis of independent mutations in the absence of natural selection. Using this and similar existing data from two other bacterial species and four eukaryotic species, we find strong evidence that deletion biases resulting in repeat contraction are common in bacteria, while eukaryotic microsatellites generally experience unbiased mutation or a bias towards insertion (repeat expansion). This difference in mutational bias suggests that eukaryotic microsatellites should generally expand wherever selection does not exclude them, whereas bacterial microsatellites should be driven to extinction by mutational pressure wherever they are not maintained by selection. This is consistent with observed bacterial and eukaryotic microsatellite distributions. Hence, mutational biases that differ between eukaryotes and bacteria can account for many of the observed differences in microsatellite DNA content and distribution found in these two groups of organisms.

Chromosome Deletion↗

PubMed2002

Effects of signal delay on auditory filter shapes derived from psychophysical tuning curves and notched-noise data obtained in simultaneous masking.

Psychophysical tuning curves (PTCs) measured in simultaneous masking usually sharpen as a short duration signal is moved from the onset to the temporal center of a longer duration masker. Filter shapes derived from notched-noise maskers have not consistently shown this effect. One possible explanation for this difference is that the signal level is fixed in the PTC paradigm, whereas the masker level is usually fixed in the notched-noise paradigm. In the present study, the signal level was fixed at 10 dB SL in both paradigms. The signal was 20 ms in duration, and presented at the onset or temporal center of the 400-ms masker. The masker was a pure tone presented in quiet (PTC) or in the presence of a pure-tone "restrictor" intended to limit off-frequency listening (PTCr), or it was a noise with a spectral notch placed symmetrically or asymmetrically about the 2-kHz signal frequency. Filter shapes were derived from the PTC, PTCr, and notched-noise data using the roex (p, w, t) model. The effects of signal delay and masking paradigm on filter bandwidth were analyzed with a two-factor repeated-measures ANOVA. There was a significant effect of signal delay (the filters sharpened with time) and masking paradigm (the filters derived from the notched-noise data were significantly wider than those derived from either of the PTC measurements, which did not differ from one another). Although the interaction between delay and paradigm was not significant, the filter derived from the notched-noise data sharpened more with time than did the other filters, and thus the bandwidth of the filters from the three paradigms were more similar at the longer delay than at the shorter delay. It is likely that the tuning-curve and notched-noise paradigms measure the same underlying filtering, but that various other factors contribute differentially to the derived filter shapes.

Adult↗

PubMed2002

Trinucleotide GAA repeats dictate pMGA gene expression in Mycoplasma gallisepticum by affecting spacing between flanking regions.

The pMGA genes of the avian respiratory pathogen Mycoplasma gallisepticum encode a family of hemagglutinins that are subject to phase variation. A trinucleotide GAA repeat region is located upstream of the pMGA transcription start site. The length of the repeat region varies at a high frequency due to changes in the number of repeat units. Previous studies have shown that pMGA genes are transcribed when 12 GAA repeats are present but are not transcribed when the number of repeats is not 12. To further analyze the mechanism of gene regulation, the pMGA promoter region was modified either by deleting the nucleotides 5" of the GAA repeats or by inserting linkers of 10 or 12 bp at a position 3" of the repeats. The modified promoter region was fused to a promoterless lacZ gene and transformed into M. gallisepticum by using transposon Tn4001 as a vector. Transformants and successive generations of progeny were analyzed with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to monitor beta-galactosidase activity. For the transformants of M. gallisepticum containing the reporter with deletion of nucleotides 5" of the GAA repeats, GAA-dependent pMGA gene regulation was abolished. For the transformants containing the reporter with an addition of 10- or 12-bp linkers, lacZ was expressed only when eight GAA repeats were present. These data indicate that the nucleotides 5" of the GAA repeats as well as the spacing between the GAA repeats and sequences downstream (3") of the repeats are important for pMGA gene expression.

5' Flanking Region↗