scholarly journals CitRWP May not be the Major or Only Major Gene that Regulates Apomixis in Citrus and Its Close Related Genus

2020 ◽  
Author(s):  
Yanhong Hou ◽  
Guizhi Gong ◽  
Zhuchun Peng ◽  
Ai Luo ◽  
Yang Cheng ◽  
...  
Keyword(s):  
Sweet Orange ◽  
Stop Codon ◽  
Major Gene ◽  
Young Leaf ◽  
Expression Level ◽  
Start Codon ◽  
Regulatory Sequence ◽  
Related Genus ◽  
Rt Pcr ◽  
Significant Difference

Abstract Background: Nucellar embryony in citrus and its close related genus is a kind of apomixis, it interferes the cross breeding and affects the genetic evolution and taxonomy in citrus. Many hypotheses have been suggested on regulation of apomixes in citrus but none of them are able to lock the true regulatory sequence until the recently reported candidate gene citRWP. However, the support evidences for the judgment of citRWP as the major or only major gene are still not very sound.Results: Firstly, M/P (Monoembryonic / Polyembryonic) marker, which was designed according to the upper stream sequence of citRWP, was used to check whether the marker exhibiting consistent segregation in different embryonic genotypes. The result showed that all sweet orange selections either monoembryonic or polyembryonic contained the P-marker, but no P-marker was detected in polyembryonic Poncirus, and a new band pattern was dectected in 9 polyembryonic progenies of W-Murcott × Flying dragon trifoliate orange. Subsequently, CitRWP expressions were analyzed with RT-PCR and Realtime RT-PCR. RT-PCR revealed that CitRWP expressed in ovule of all varieties. Realtime RT-PCR results in ovule were consistent with RT-PCR. The expression value of monoembryonic pummelo was the lowest, and there was no significant difference between polyembroynic and monoembryonic sweet orange cultivars. In the hybrid offsprings, the expression of the CitRWP gene showed an extreme two-stage differentiation. The expression in young leaf and ovule of polyembryonic and monoembryonic sweet orange cultivars were different, with a higher expression level in young leaf and a lower expression level in ovule of polyembryonic cultivars. Finally, the cDNA sequences of CitRWP from ovules were cloned and sequenced. Results revealed that the CitRWP has great variations in Poncirus, which included SNPs in start-codon and/or stop-codon as well as fragment insertion/deletion causing incorrect translation. The monoembryonic/polyembryonic sweet orange cultivars and pummelo varieties had similar variations also.Conclusion: CitRWP were not consistently distributed and expressed in different embryonic genotypes. It may not be the major or only major gene that regulates apomixis in citrus and its close related genus.

5′-Nucleosidase and Deoxycytidine Kinase Gene Expressions in High-Risk Myelodysplastic Syndrome (MDS).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3434-3434
Author(s):  
Keijiro Suzuki ◽  
Takeshi Sugawara ◽  
Yoji Ishida
Keyword(s):  
Overall Survival ◽  
High Risk ◽  
Clinical Outcome ◽  
Mrna Expression ◽  
Expression Level ◽  
Deoxycytidine Kinase ◽  
Rt Pcr ◽  
Significant Difference ◽  
The Mean ◽  
Classification Quantitative

Abstract Various chemotherapeutic regimens including cytarabine (ara-C) for MDS patients have been less effective than those for AML patients. Althogh alterations in the expression of genes involved in ara-C metabolism have been reported to be related to the clinical outcome in AML patients, there is no report about MDS. Deoxycytidine kinase (dCK) is the rate-limiting enzyme in this activation process being the first phosphorylation of ara-C. Cytoplasmic 5′-nucleotidase (5′-NT) dephosphorylates ara-CMP. The concentration of the intracellular ara-CTP, active form of ara-C, depends on the activities of two enzymes. To determine whether the level of expression of 5′-NT and dCK is implicated in clinical outcome in patients with high-risk MDS (RAEB/RAEB-t), we analyzed the mRNA expression of these at diagnosis in bone marrow (BM) cells of patients with RAEB/RAEB-t using real-time PCR (rt-PCR). Materials and Methods: BM cells obtained from 22 patients (male: female=16:6) with untreated high-risk MDS. The mean age of all patients was 66.4±7.9 y. 7 patients (31.8%) had RAEB-t subtype and 15 (68.2%) patients RAEB subtype of the FAB classification. Quantitative rt-PCR of 5′-NT or dCK mRNA of BM cells in patients and 12 healthy volunteers was performed by LightCycler using β-actin as housekeeping control. For each sample, a ratio of 5′-NT or dCK value/β-actin value was calculated and considered as the level of 5′-NT or dCK mRNA expression. Results: 15 of patients received ara-C containing regimen (7 patients had supported care only). Complete remission was obtained in 7 patients. Other 8 patients were chemoresistant. The median overall survival (OS) of all patients was 22.9 months (range 2.0–91.0), and that of patients with chemotherapy was 20.0 (7–91). The median post-chemotherapy survival (PCS) was 15.0 months (5.0–43.0). At diagnosis, the mean and median expression level of 5′-NT mRNA in all patients was 1.56 (SD 1.61) and 1.36 (range 0.77–6.77), respectively (control: mean 0.23, SD 0.06 and median 0.67, range: 0.17–0.34, p<0.01). The expression of dCK mRNA did not show any significant difference between patients and control. Patients with chemotherapy whose BM cells were higher level of 5′-NT expression (greater than mean value) had shorter OS (14.0 months vs. 26.0 months, p<0.01) and shorter PCS (10.0 months vs. 17.0 months, p=0.011). Figure Figure Conclusion: These data suggest that the expression level of 5′-NT mRNA might be a candidate for prognostic factor in RAEB/RAEB-t. Patients with an increase of 5′-NT mRNA at diagnosis show shorter overall survival and chemoresistance.

Prognostic Significance Of CRLF2 Over-Expression In Pediatric Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1368-1368
Author(s):  
Mio Yano ◽  
Toshihiko Imamura ◽  
Daisuke Asai ◽  
Akiko Moriya Saito ◽  
So-ichi Suenobu ◽  
...  
Keyword(s):  
Prognostic Factor ◽  
Prognostic Significance ◽  
Fusion Transcript ◽  
Genetic Alterations ◽  
Expression Level ◽  
Rt Pcr ◽  
Poor Prognostic Factor ◽  
Over Expression ◽  
Significant Difference ◽  
The Impact

Abstract Background Previous studies have reported that IKZF1 deletion was an adverse prognostic factor in pediatric B-cell-precursor ALL (BCP-ALL). However, the prognostic significance of CRLF2 over-expression (OE) is controversial. In order to assess the prognostic value of CRLF2 OE and genetic alterations involving CRLF2, we conducted genetic analysis of CRLF2 in pediatric BCP-ALL treated according to Japan Association of Childhood Leukemia Study (JACLS) ALL02 cohort. Methods We examined diagnostic bone marrow or peripheral blood samples of 167 pediatric BCP-ALL patients treated on the JACLS ALL02 protocol. The analyzed cohort included 68 patients classified in NCI-SR and 99 patients in NCI-HR. The deletion of IKZF1 and gain of CRLF2 were determined by MLPA. CRLF2 expression level was determined by quantitative RT-PCR and OE was defined as 10 times more than mean expression level of 167 cases. The P2RY8-CRLF2 fusion was detected by RT-PCR. To identify fusion transcript related to CRLF2 OE, we performed messenger RNA sequencing (mRNA-seq) on six of 17 patients with CRLF2 OE without CRLF2 gain and P2RY8-CRLF2 fusion. Results CRLF2 OE was identified in 30 (18%) of 167 patients, which was similar to that reported previously. IKZF1 deletion was found in 25(15%) of 167 patients, which was found more in CRLF2 OE patients than non-CRLF2 OE patients (30% vs 11%, p<0.05). P2RY8-CRLF2 fusion was identified in only 3 (1.7%) of 167 patients, and all of them were classified in CRLF2 OE. In detail, only one of three P2RY8-CRLF2 positive patients had IKZF1 deletion. One of two P2RY8-CRLF2 positive patients without IKZF1 deletion harbored MLL-AF4 fusion. CRLF2 gain was identified in 18(11%) of 167 patients. Eleven of these 18 patients were classified in CRLF2 OE, suggesting CRLF2 gain was significantly related to CRLF2 OE (37% vs 5%, p<0.01). Interestingly, none of CRLF2 OE patients with CRLF2 gain did have IKZF1 deletion. We identified one patient with novel fusion transcript caused by the 32kb deletion from CRLF2 intron 1. This patient was classified in CRLF2 OE with IKZF1 deletion, suggesting that the novel fusion transcript was related to CRLF2 OE. However, we could not identify any fusion transcripts related to CRLF2, such as IgH@-CRLF2, in remaining five patients by mRNA-seq. Interestingly, we identified three EBF1-PDGFRb positive patients in CRLF2 OE patients with IKZF1 deletion who did not have genetic alterations including CRLF2. In survival analysis, significant difference on the 5-year event-free survival (EFS) and overall survival (OS) between patients with and without CRLF2 OE was not observed (71% vs 75%, log rank p=0.68, 96% vs 82%, log rank p=0.11). In addition, type of genetic alterations related to CRLF2, such as CRLF2 gain and P2RY8-CRLF2 fusion, did not show the impact on EFS and OS in CRLF2 OE patients. However, significant difference on 5-year EFS between CRLF2 OE patients with and without IKZF1deletion was observed (44% vs 83%, log rank p=0.02). In multivariate analysis of 167 patients, only IKZF1 deletion was significant predictors of inferior OS (Hazard ratio: 2.427, 95%CI:1.037-5.679, p=0.04). Discussions The current analysis revealed that CRLF2 OE was caused by several different mechanisms, such as CRLF2 gain, P2RY8-CRLF2 fusion and rare fusion transcript related to CRLF2. However, regardless of type of genetic alterations of CRLF2, concomitant IKZF1 deletion holds the major impact on poor prognosis in CRLF2 OE patients. Especially, CRLF2 gain and IKZF1 deletion were mutually exclusive. Thus, CRLF2 OE caused by CRLF2 gain was not poor prognostic factor for high risk BCP-ALL. The prognostic significance of CRLF2 should be evaluated in consideration of IKZF1 status. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Latanoprost eye drops induce conjunctival lymphatic vessel development

2021 ◽  
Vol 14 (9) ◽  
pp. 1345-1349
Author(s):  
Kai Ma ◽  
◽  
Cheng-Juan Yin ◽  
Zhen-Yong Zhang ◽  
◽  
...  
Keyword(s):  
Expression Level ◽  
Control Group ◽  
Eye Drops ◽  
Rt Pcr ◽  
Protein Expression Level ◽  
Significant Difference ◽  
New Zealand White Rabbits ◽  
Polymerase Chain ◽  
Vessel Development ◽  
And Control

AIM: To investigate the effect of latanoprost eye drops on the conjunctival lymphatics. METHODS: Twenty-four healthy New Zealand White rabbits weighing 1.5 to 2.0 kg were randomly divided into three groups: latanoprost group (n=8) administered with latanoprost eye drops once a day for 2mo, carteolol group (n=8) administered with carteolol eye drops once a day for 2mo, and control group (n=8) without any treatment. The conjunctival tissues in the three groups were extracted to investigate the expression levels of 5’-nucleotidase (5’-Nase) by Western blot, reverse transcription-polymerase chain reaction (RT-PCR), and immunofluorescence staining, respectively. RESULTS: The protein expression level of 5’-Nase was significantly higher in latanoprost group than carteolol group (F=231.175, P<0.001) and control group (P<0.001), while there was no significant difference between the carteolol group and the control group (P>0.05). The mRNA expression level of 5’-Nase in the latanoprost group was also significantly higher than carteolol group (F=71.169 P<0.005) and control group (P<0.005). The conjunctival lymphatics were positive immunofluorescence stained with the 5’-Nase antibodies in the latanoprost group and not stained in the control group. CONCLUSION: Latanoprost eye drops can induce conjunctival lymphangiogenesis which may be concerned in clinical implications.

scholarly journals RT-PCR analysis of Deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor)

2005 ◽  
Vol 86 (12) ◽  
pp. 3419-3424 ◽  
Author(s):  
Constanze Yue ◽  
Elke Genersch
Keyword(s):  
Varroa Destructor ◽  
Pcr Analysis ◽  
Body Parts ◽  
Rt Pcr ◽  
Larval Food ◽  
Viral Pathogen ◽  
Deformed Wing Virus ◽  
Significant Difference ◽  
Almost All ◽  
Viral Sequences

Deformed wing virus (DWV) is a honeybee viral pathogen either persisting as an inapparent infection or resulting in wing deformity. The occurrence of deformity is associated with the transmission of DWV through Varroa destructor during pupal stages. Such infections with DWV add to the pathology of V. destructor and play a major role in colony collapse in the course of varroosis. Using a recently developed RT-PCR protocol for the detection of DWV, individual bees and mites originating from hives differing in Varroa infestation levels and the occurrence of crippled bees were analysed. It was found that 100 % of both crippled and asymptomatic bees were positive for DWV. However, a significant difference in the spatial distribution of DWV between asymptomatic and crippled bees could be demonstrated: when analysing head, thorax and abdomen of crippled bees, all body parts were always strongly positive for viral sequences. In contrast, for asymptomatic bees viral sequences could be detected in RNA extracted from the thorax and/or abdomen but never in RNA extracted from the head. DWV replication was demonstrated in almost all DWV-positive body parts of infected bees. Analysing individual mites for the presence of DWV revealed that the percentage of DWV-positive mites differed between mite populations. In addition, it was demonstrated that DWV was able to replicate in some but not all mites. Interestingly, virus replication in mites was correlated with wing deformity. DWV was also detected in the larval food, implicating that in addition to transmission by V. destructor DWV is also transmitted by feeding.

Biochemical parameters and relation to disease severity in COVID-19 patients

2021 ◽  
pp. 93-96
Keyword(s):  
Biochemical Parameters ◽  
Troponin I ◽  
Total Bilirubin ◽  
Early Stage ◽  
Length Of Hospital Stay ◽  
Rt Pcr ◽  
Sodium Potassium ◽  
Appropriate Treatment ◽  
Significant Difference ◽  
Severity Of The Disease

Aim: In this study, we aimed to evaluated whether there is an association between the biochemistry parameters obtained from the first blood test after hospitalization of COVID 19 patients and the prognosis and severity of the disease. Thus, we planned to identify patients with a severe course at an early stage and to help physicians determine the appropriate treatment. Material and Method: The study included 106 COVID 19 patients confirmed by RT-PCR. Patients were categorized into two groups: those admitted to the hospital ward and discharged with recovery (mild cases) and those admitted directly or eventually to the intensive care unit (severe cases). Biochemical parameters of the groups were compared with the Mann Whitney-U Test, as none of the compared parameters fit the normal distribution. Results: There was no statistically significant difference between the male-female numbers and ages of the two groups. Statistically significant differences were observed in the length of hospital stay, procalcitonin, hs-troponin I, ferritin, glucose, urea, creatinine, calcium, direct bilirubin, AST, LDH and CRP values (p<0,05). However, no significant difference was found in sodium, potassium, chloride, total bilirubin and ALT tests. Conclusion: The results show that some biochemistry parameters may be used to predict the prognosis of the disease. In particular, procalcitonin, hs troponin I, LDH and CRP values seem to be moderate biomarkers of the prognosis of the disease.

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression

1987 ◽  
Vol 7 (8) ◽  
pp. 2914-2924
Author(s):  
A Hoekema ◽  
R A Kastelein ◽  
M Vasser ◽  
H A de Boer
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Codon Usage ◽  
Experimental Approach ◽  
Mrna Translation ◽  
Yeast Cells ◽  
Expression Level ◽  
Start Codon ◽  
Mrna Levels

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.

Comparison of ELISA and RT-PCR assays for the detection of Tomato spotted wilt virus in peanut

2009 ◽  
Vol 36 (2) ◽  
pp. 133-137 ◽  
Author(s):  
P. M. Dang ◽  
D. L. Rowland ◽  
W. H. Faircloth
Keyword(s):  
Tomato Spotted Wilt Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Infection Rates ◽  
Rt Pcr ◽  
Chi Square ◽  
Tomato Spotted Wilt ◽  
Significant Difference ◽  
Pcr Assays ◽  
Chi Square Analysis ◽  
Wilt Virus

Abstract Diagnosis of Tomato spotted wilt virus (TSWV) in peanut can be accomplished by enzyme-linked immunosorbent assay (ELISA) or reverse transcription polymerase chain reaction (RT-PCR) but there has been no report of a direct comparison of the success of the two assays in evaluating infection rates of field-grown peanut. We collected peanut root samples from field-grown plants, 76 in 2006 and 48 in 2007, and tested these samples by both ELISA and RT-PCR assays for the presence of TSWV. Out of 124 samples, 50 (40.3%) and 57 (46.0%) were positive for TSWV by ELISA and RT-PCR respectively. In 13.7% of these samples, ELISA and RT-PCR differed in their results. However, Chi square analysis showed no significant difference between the results for these two assays. This result supports the conclusion that ELISA and RT-PCR are comparable for detecting TSWV infection rates in field-grown peanuts.

scholarly journals Effects of L-655,708 on expression changes of GABA, glutamate, and beta-endorphin induced by propofol anesthesia in rats

2018 ◽  
Vol 16 ◽  
pp. 205873921879670
Author(s):  
Jin Wang ◽  
Xinyi Li ◽  
Huisheng Wu ◽  
Jianjuan Ke ◽  
Zongze Zhang ◽  
...  
Keyword(s):  
Dentate Gyrus ◽  
Cognitive Dysfunction ◽  
Sham Group ◽  
Postoperative Cognitive Dysfunction ◽  
Expression Level ◽  
Control Group ◽  
Beta Endorphin ◽  
Anesthetized Rats ◽  
Significant Difference ◽  
Dentate Gyrus Region

Anesthetics are considered to be one of the important inducing factors of postoperative cognitive dysfunction (POCD). The hippocampal region of the rat is one of the action sites of general anesthesia drugs. L 655,708, a reverse agonist of gamma aminobutyric acid (GABA) receptor, can significantly improve short-term memory dysfunction in mice after anesthetized with isoflurane. So the purpose of this study is to investigate the effects of L-655,708 on expression of GABA, glutamate (GLU), and beta-endorphin (β-EP) in the dentate gyrus region of the hippocampus and cognition of rats anesthetized with propofol. In all, 30 male Sprague–Dawley (SD) rats were randomly allocated into the control group, sham group, and L-655,708 group, with 10 in each group. The cognitive function of rats was measured by Morris water maze before and 1 h after administration. Then the rats were sacrificed for brain tissues. Immunohistochemistry was used to study the expression of GABA, GLU, and β-EP in the hippocampus of anesthetized rats in each group. Compared with the control group, the latency of the sham group and L-655,708 group were significantly prolonged after administration ( P < 0.05). However, L-655,708 could shorten the prolonged latency ( P < 0.05). There was no significant difference in times of accessing original platform area between the three groups before and after medication ( P > 0.05). The expression level of GABA in the dentate gyrus region of hippocampus of rats in the sham group was significantly higher than that in the control group ( P < 0.05), while the expression level in the L-655,708 group was significantly lower than that in the sham group ( P < 0.05). No significant difference was found in the expression of GLU in the dentate gyrus region of hippocampus of rats in each group ( P > 0.05). Compared with the control group, the expression of β-EP was significantly lower in the dentate gyrus region of the hippocampus of sham group rats ( P < 0.05). However, the expression of β-EP in the L-655,708 group was significantly higher than that in the sham group ( P < 0.05). Cognitive dysfunction in rats anesthetized with propofol may be related to high expression of GABA and low expression of β-EP in the hippocampus. The mechanism of L-655,708 in reducing the cognitive impairment in propofol anesthetized rats may be bound up with down-regulating the expression of GABA and increasing the expression of β-EP in the hippocampus.

scholarly journals The Neuroprotective Effects of Xenon on Neonatal Rats with White Matter Damage by Regulating Expression of microRNA-210 and HIF-1αThe Neuroprotective Effects of Xenon on Neonatal Rats with White Matter Damage by Regulating Expression of microRNA-210 and HIF-1α

2020 ◽  
Author(s):  
Xiangyun Yin ◽  
Jixiu Zhao ◽  
Jian Jiang ◽  
Hongmin Xi ◽  
Xianghong Li ◽  
...  
Keyword(s):  
White Matter ◽  
Expression Level ◽  
Neonatal Rats ◽  
Rt Pcr ◽  
White Matter Damage ◽  
Neuroprotective Effects ◽  
Brain White Matter ◽  
Group A ◽  
Group B ◽  
Brain Tissues

Abstract Background:Premature infant is a significant health care burden. White matter damage (WMD) is a leading cause of acute mortality and chronic morbidity in preterm. Xenon (Xe) intervention was given to the 3-day-old neonatal rats with brain white matter injury. By detecting the changes in the expression level of microRNA210 and hypoxia inducible factor 1α (HIF-1α) in brain tissue before and after xenon intervention, we can research the molecular basis and the mechanism of neuroprotective on effect of xenon on brain white matter damage in neonatal rats.Methods:Three-day-old SD rats were randomly divided into sham group(Group A, n=24), lipopolysaccharide(LPS)+hypoxia-ischemia(HI) group (Group B, n=24) and LPS+HI+Xe group ( n=72). The onset of Xe inhalation started at 0,2 and 5 hours in subgroups C,D,and E respectively.We investigated the neurobehavioral deficits by performing TUNEL and hematoxylin and eosin (HE) staining and examining the expression of miR-210and HIF-1α in brain tissues via RT-PCR and western blot. Results: Xe treatment improved the histological alterations and decreased the number of apoptotic cells in group C pups.Compared to group A,Detection of miR-210 level by RT-PCR. the expression level of miR-210 in neonatal rats' periventricular tissue increased significantly at all time points in group B (p<0.05).While the expression level of miR-210 in brain tissues of group B was significantly lower at 48h and 72h than that of group C(p<0.05).Similarly,Detection of HIF-1α protein by Western blot. The level of HIF-1α protein in group B brain tissues was significantly higher than that of group A at each time point (p<0.05), Xe treatment resulted in a marked increase in HIF-1α in C,D, and E subgroups (P < 0.05, compared to group B).Conclusions: These results demonstrate that the expression of HIF-1α and miR-210 increased in periventricular tissues and Xe could relieve the white matter damage by up-regulating the expression of HIF-1α and its target gene miR-210.The Xe therapeutic time window was within 5 hours after intervention, the sooner the better.

scholarly journals Translationskopplung via Termination-Reinitiation in Archaea und Bacteria

2021 ◽  
Author(s):  
◽  
Madeleine Huber
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Stop Codon ◽  
Haloferax Volcanii ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Directed Mutagenesis ◽  
Ribosome Display ◽  
E Coli

Operons wurden zuerst im Jahre 1961 beschrieben. Bis heute ist bekannt, dass die prokaryotischen Domänen Bacteria und Archaea Gene sowohl in monocistronischen als auch in bi- oder polycistronischen Transkripten exprimieren können. Häufig überlappen Gene sogar in ihren Sequenzen. Diese überlappenden Genpaare stehen nicht in Korrelation mit der Kompaktheit ihres Genoms. Das führt zu der Annahme, dass eine Art der Regulation vorliegt, welche weitere Proteine oder Gene nicht benötigt. Diese könnte eine gekoppelte Translation sein. Das bedeutet die Translation des stromabwärts-liegenden Gens ist abhängig von der Translation eines stromaufwärts-liegenden Gens. Diese Abhängigkeit kann zum Beispiel durch lang reichende Sekundärstrukturen entstehen, bei welchen Ribosomenbindestellen (RBS) des stromabwärts-liegenden Gens blockiert sind. Die de novo-Initiation am stromabwärts-liegenden Gen kann nur stattfinden, wenn das erste Gen translatiert wird und dabei die Sekundärstruktur an der RBS aufgeschmolzen wird. Für Genpaare in E. coli ist dieser Mechanismus gut untersucht. Ein anderes Beispiel für die Translationskopplung ist die Termination-Reinitiation, bei welcher ein Ribosom das erste Gen translatiert bis zum Stop-Codon, dort terminiert und direkt am stromabwärts-liegenden Start-Codon reinitiiert. Der Mechanismus via Termination-Reinitiation ist bis jetzt nur für eukaryontische Viren beschrieben worden. Im Gegensatz zu einer Kopplung über Sekundärstrukturen kommt es bei der Termination-Reinitiation am stromabwärts-liegenden Gen nicht zu einer de novo-Initiation sondern eine Reinitiation des Ribosoms findet statt. Diese Arbeit analysiert jene Art der Translationskopplung an Genen polycistronischer mRNAs in jeweils einem Modellorganismus als Vertreter der Archaea (Haloferax volcanii) und Bacteria (Escherichia coli). Hierfür wurden Reportergenvektoren erstellt, welche die überlappenden Genpaare an Reportergene fusionierten. Für diese Reportergene ist es möglich die Transkriptmenge zu quantifizieren sowie für die exprimierten Proteine Enzymassays durchgeführt werden können. Aus beiden Werten können Translationseffizienzen berechnet werden indem jeweils die Enzymaktivität pro Transkriptmenge ermittelt wird. Durch ein prämatures Stop-Codon in diesen Konstrukten ist es möglich zu unterscheiden ob es für die Translation des zweiten Gens essentiell ist, dass das Ribosom den Überlapp erreicht. Hiermit konnte für neun Genpaare in H. volcanii und vier Genpaare in E. coli gezeigt werden, dass eine Art der Kopplung stattfindet bei der es sich um eine Termination-Reinitiation handelt. Des Weiteren wurde analysiert, welche Auswirkungen intragene Shine-Dalgarno Sequenzen bei dem Event der Translationskopplung besitzen. Durch die Mutation solcher Motive und dem Vergleich der Translationseffizienzen der Konstrukte, mit und ohne einer SD Sequenz, wird für alle analysierten Genpaare beider Modellorganismen gezeigt, dass die SD Sequenz einen Einfluss auf diese Art der Kopplung hat. Zwischen den Genpaaren ist dieser Einfluss jedoch stark variabel. Weiterhin wurde der maximale Abstand zwischen zwei bicistronischen Genen untersucht, für welchen Translationskopplung via Termination-Reinitiation noch stattfinden kann. Hierfür wird durch site-directed mutagenesis jeweils ein prämatures Stop-Codon im stromaufwärts-liegenden Gen eingebracht, welches den intergenen Abstand zwischen den Genen in den jeweiligen Konstrukten vergrößert. Der Vergleich aller Konstrukte eines Genpaars zeigt in beiden Modellorganismen, dass die Termination-Reinitiation vom intergenen Abstand abhängig ist und die Translationseffizienz des stromabwärts-liegenden Reporters bereits ab 15 Nukleotiden Abstand abnimmt. Eine weitere Fragestellung dieser Arbeit war es, den genauen Mechanismus der Termination-Reinitiation zu analysieren. Für Ribosomen gibt es an der mRNA nach der Termination der Translation zwei Möglichkeiten: Entweder als 70S Ribosom bestehen zu bleiben und ein weiteres Start-Codon auf der mRNA zu suchen oder in seine beiden Untereinheiten zu dissoziieren, während die 50S Untereinheit die mRNA verlässt und die 30S Untereinheit über Wechselwirkungen an der mRNA verbleiben kann. Um diesen Mechanismus auf molekularer Ebene zu untersuchen, wird ein Versuchsablauf vorgestellt. Dieser ermöglicht das Event bei der Termination-Reinitiation in vitro zu analysieren. Eine Unterscheidung von 30S oder 70S Ribosomen bei der Reinitiation der Translation des stromabwärts-liegenden Gens wird ermöglicht. Die Idee dabei basiert auf einem ribosome display, bei welchem Translationskomplexe am Ende der Translation nicht in ihre Bestandteile zerfallen können, da die eingesetzte mRNA kein Stop-Codon enthält Der genaue Versuchsablauf, die benötigten Bestandteile sowie proof-of-principal Versuche sind in der Arbeit dargestellt und mögliche Optimierungen werden diskutiert.

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