Academic literature on the topic 'Differential display polymerase chain reaction amplification'

Investigators approaching the problem of renal organogenesis have been hampered by a paucity of suitable molecular markers that specify distinct developmental phenotypes. To identify such markers, differential display-polymerase chain reaction (DD-PCR) was used to survey the temporal pattern of gene expression in mouse kidney at 11.5, 13.5, 15.5, and 17.5 days after conception and in the adult kidney. Twenty-two differentially expressed amplification products were identified, isolated, and sequenced. Seventeen clones showed no significant similarity with previously reported nucleotide sequences: two were similar to two housekeeping gene products, and three were similar to human or rat expressed sequence tags. To confirm the differential expression patterns observed by DD-PCR, semiquantitative reverse transcription-PCR was performed using sequence-specific oligonucleotide primers. Nineteen of 22 clones were differentially expressed during kidney development [mouse embryonic renal marker (MERM) sequences 1-19]. The value of MERMs as developmental markers was further assessed in mouse metanephric organ culture, where the pattern of MERM transcript expression mimicked that observed in vivo. Therefore, the DD-PCR method permitted development of a panel of marker sequences that can be used to characterize renal developmental processes and that may allow the identification of novel, functionally relevant gene products.

Abstract The mRNA differential display technique was performed to investigate the differences in gene expression in the Longissimus dorsi muscle tissues from Landrace×Large White cross-combination. One novel gene that was differentially expressed was identified using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and its complete cDNA sequence was obtained using the rapid amplification of cDNA ends (RACE) method. The nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 260 amino acids that contains the putative conserved domain of the carbonic anhydrase, and this protein has high homology with the carbonic anhydrase III (CA-III) of four species-mouse (91%), horse (91%), rat (89%) and human (86%)–so that it can be defined as swine carbonic anhydrase III. The phylogenetic tree analysis revealed that the swine CA-III has a closer genetic relationship with the horse CA-III than with those of mouse, rat and human. The tissue expression analysis indicated that the swine CA-III gene is generally expressed in most tissues. Our experiment is the first to establish the primary foundation for further research on the swine CA-III gene.

Avirulence (AVR) genes in Magnaporthe oryzae, the fungal pathogen that causes the devastating rice blast disease, have been documented to be major targets subject to mutations to avoid recognition by resistance (R) genes. In this study, an AVR-gene-based diagnosis tool for determining the virulence spectrum of a rice blast pathogen population was developed and validated. A set of 77 single-spore field isolates was subjected to pathotype analysis using differential lines, each containing a single R gene, and classified into 20 virulent pathotypes, except for 4 isolates that lost pathogenicity. In all, 10 differential lines showed low frequency (<24%) of resistance whereas 8 lines showed a high frequency (>95%), inferring the effectiveness of R genes present in the respective differential lines. In addition, the haplotypes of seven AVR genes were determined by polymerase chain reaction amplification and sequencing, if applicable. The calculated frequency of different AVR genes displayed significant variations in the population. AVRPiz-t and AVR-Pii were detected in 100 and 84.9% of the isolates, respectively. Five AVR genes such as AVR-Pik-D (20.5%) and AVR-Pik-E (1.4%), AVRPiz-t (2.7%), AVR-Pita (0%), AVR-Pia (0%), and AVR1-CO39 (0%) displayed low or even zero frequency. The frequency of AVR genes correlated almost perfectly with the resistance frequency of the cognate R genes in differential lines, except for International Rice Research Institute-bred blast-resistant lines IRBLzt-T, IRBLta-K1, and IRBLkp-K60. Both genetic analysis and molecular marker validation revealed an additional R gene, most likely Pi19 or its allele, in these three differential lines. This can explain the spuriously higher resistance frequency of each target R gene based on conventional pathotyping. This study demonstrates that AVR-gene-based diagnosis provides a precise, R-gene-specific, and differential line-free assessment method that can be used for determining the virulence spectrum of a rice blast pathogen population and for predicting the effectiveness of target R genes in rice varieties.

The cell wall acts as the first line of defense during pathogen invasion. Polygalacturonases (PGs) are a class of cell-wall-modifying enzymes with precise temporal and organ-specific expression. A 350-bp fragment with high homology to PGs was identified by differential display (DD) analysis of soybean cyst nematode (SCN) race 3 resistant PI 437654 and susceptible cultivar Essex. The fragment was strongly expressed in Essex, 2 days after inoculation (DAI). Complete coding sequences of two PG cDNAs, PG1 and PG2, were isolated by 3′ and 5′ rapid amplification of cDNA ends polymerase chain reaction (RACE PCR). PI 437654 and Essex had identical PG1 and PG2 sequences. A transversion from A to C created a PstI restriction site in the PG2 cDNA that was used to distinguish the two PG cDNAs by cleaved amplified polymorphic sequence (CAPS) analysis. A cDNA encoding a polygalacturonase-inhibitor protein (PGIP) that is 89% identical to the Phaseolus vulgaris PGIP was isolated from soybean roots by reverse transcription (RT)-PCR. Steady-state levels of PG and PGIP were investigated by RNA gel blot analysis in roots 1 to 5 DAI and in hypocotyls and leaves. Differences in the constitutive levels of PG mRNAs were observed in roots of different soybean genotypes. Steady-state levels of PG mRNAs were enhanced during compatible interactions with SCN and reduced in incompatible interactions and in mechanically wounded roots. Enhanced PGIP transcription was observed in response to mechanical wounding in both PI 437654 and Essex, but only in compatible interactions with SCN, suggesting uncoupling of PGIP functions in developmental and stress cues. Constitutive expression in incompatible interactions shows PGIP is not a factor in SCN resistance. Thus, the up-regulation of endogenous PG transcription in soybean roots early after SCN infection could facilitate successful parasitism by SCN.

Nucleic acid aptamers are a kind of one-dimensional biological nanomaterials and have found many applications. This paper designed and implemented a polymerase chain reaction (PCR) amplification device with a reaction volume of 500 μL, which can be used for the amplification of nucleic acid aptamers of tumor cells in the aptamer selection. This device mainly includes a control module, a temperature measuring module, a PCR amplification tube, a metal tank module, a liquid crystal display (LCD) and operation module and a cooling module. The new PCR amplification chamber is matched with the designed metal tank to ensure the temperature uniformity of the PCR amplification solution. The control module based on the STM32F103RCT6 manages the workflow of the entire device. The PCR amplification chamber and PT100 sensors on the metal tank formed a closed-loop feedback system, and the incremental proportional-integral-derivative (PID) algorithm was used to achieve the precise temperature control. In addition, we introduced the Smith predictive compensation algorithm to solve the temperature hysteresis problem of the PCR amplification chamber. The experimental results showed that the PCR device can meet the requirements for the nucleic acid aptamer selection of tumor cells. The device can also be used in other experiments with large-volume PCR amplification.

Despite significant advances in diagnosis and treatment, breast cancer remains the leading cause of cancer-related deaths in Australian women. Colorectal cancer is the second most common cancer in both males and females; after prostate and breast cancer, respectively, and excluding non-melanocytic skin cancer. Both breast cancer and colorectal cancer follow a common progressive course of illness; presenting (at least initially) with benign symptoms that can be treated by ablation (or removal) of the affected area. Cancer progression is associated with breakdown of tissue barriers (such as basement membranes), leading to the spread of cancer cells (via the vasculature or lymphatic system), and the establishment of secondary metastatic disease at green-field sites. Secondary tumours presenting in the lungs, ovaries, liver, bone, or brain are associated with chronic-debilitating symptoms that are difficult to treat, and will result in death. In the case of breast and colon cancer, effective early therapeutic intervention does have a significant impact upon patient survival. Tumour progression in breast and colon carcinomas is characterised by invasion of the surrounding stroma, and the acquisition of stromal characteristics, by previously epithelial cells. This progression is associated with the expression of extracellular proteases (ECPs) and increased motility. The process of mesenchymal transformation that tumour cells undergo is also referred to as the epithelial to mesenchymal transition (EMT). In general terms the aim of the study, presented in this thesis, was to investigate gene expression in cancer biology; and to characterise changes in breast cancer and colon cancer, with a focus on those genes, and gene products that may play a role in metastasis, including a family of ECPs, the matrix metalloproteinases (MMPs). In our laboratory, we have applied methods in microdissection, differential display polymerase chain reaction amplification (DD-PCR), and array hybridisation analysis to identify gene expression patterns in late stage archival formalin fixed paraffin embedded (FFPE) breast tumour biopsies that may be indicative of the EMT; or the response to the surrounding stroma/interstitium to the presence of the tumour.' The quality of nucleic acid obtainable from FFPE material presents a considerable challenge for gene expression studies. In order to identify tissue specific gene expression patterns, DD-PCR products, amplified from message obtained following segregation of tumour tissue from surrounding stroma, was hybridised to arrayed cDNA libraries created from stromal tumours, or sarcomas. In this way, 21 known genes, or expressed sequence tags (ESTs), were identified. These included the cytoskeletal element and EMT marker, vimentin, the mammary developmental factor and, signal transducer and activator of transcription (STAT)-3, and the cargo selection protein (TIP47). Seventeen genes showed differential expression in either the tumour, or stromal fractions. When applied to transformed breast cancer cell lines (MDA-MB-435 & T47D) DD-array analysis revealed a further 17 genes that were differentially regulated in invasive cells, compared with those displaying a less invasive phenotype. Six of the ESTs identified by DD-PCR array analysis, had no known (or predicted) function. For example, bcaf-2 was identified as the 3'-end of a putative open reading frame (ORF) localised to chromosome 6, while bcaf-10 showed homology with a known ORF. In order to analyse the expression of these bcafs further, a stromal cell culture model, representative of the original osteosarcoma cDNA libraries from which they were obtained, was used. In this model, CD14' (or adherent) peripheral blood mononuclear cells (PBMCs) treated with macrophage colony stimulating factor (M-CSF), can be allowed to differentiate into macrophage-like (ML) cells; while cells treated with M-CSF, and the receptor activator of NF-KB ligand (RANKL) will differentiate into multinucleate osteoclast-like (OCL) multinucleate giant cells. Uniquely, the stromal EST, bcaf-2 was expressed only by RANKL-treated (or OCL) cells. bcaf-2 and other ESTs, identified by DD-PCR analysis (and recently published) are the subject of on going research in our laboratory. The role of RANKL in mammary gland development and bone metastasis suggested that the identification of a RANKL-regulated stromal factor in breast tissue (bcaf-2) was not an artefact. RANKL is a membrane-bound, member of the tumour necrosis factor (TNF)-a cytokine super family. In order to test the hypothesis that RANKL might act as an inflammatory cytokine to regulate clinically significant stromal gene expression in the breast, we employed quantitative real time PCR analysis to examine the relative levels of selected members of a group of metal dependent ECPs, the matrix metalloproteinases (MMPs). RNA was extracted from ML cells and OCL cells, as well as RANK positive breast cancer cell lines (T47D, MDA-MB-435 & MCF-7). When the relative levels of protease mRNA were compared we demonstrated a significant (>20- fold) specific increase in collagenase (collagenase 2lMMP-8 and collagenase 3lMMP-13), and the tissue inhibitor of MMP (TIMP)-2 expression in M-CSF and RANKL treated PBMCs cells. When the assay was applied to RANKL treated breast cancer cell lines (MCF-7, T47D & MDAMB- 231), minor (40-fold) but potentially significant alterations in stromal protease gene expression were observed. The changes observed did not however, support the hypothesis that RANKL might act as an inflammatory cytokine to induce significant alterations in ECP expression in breast cancer cells. To investigate the role of RANKL as a driver of EMT in aberrant breast epithelium, total message (mRNNcDNA) from T47D, MCF-7, MDA-MB-231 cells, and message from the same cell lines treated with RANKL were compared by comparative fluorescent cDNA microarray analysis. Of the 1,700 targets available on the arrays, this study identified 160 that were differentially expressed in RANKL treated cells. The results suggest that RANKL may promote rather than suppress a mammary epithelial phenotype in breast cancer. In fact a putative mesenchymal to epithelial transition (MET) was observed following microscopic analysis, and this finding is the subject of on going research in our laboratory. Sporadic structural alterations in certain mitogenic factors represent important early events in cancer progression, while inherited mutations govern familial susceptibility to disease. In colon cancer, a close link exists between Winglessllnt (WNT) signalling, disease pathology, and the expression of MMPs. To examine the relationship between protease expression and structural genetic alterations in this EMT-linked signalling pathway, and others, we applied combined QPCR analysis of MMP expression and PCR-Single Strand Conformation Analysis (SSCA) to 26 colonic tumours, and patient-matched normal colonic mucosa. In this study, significant correlations between the expression of ECPs, and a key mediator of WNT signalling (p-catenin) were identified. While tumours possessing specific functional mutations in K-Ras, were found to group with phenotypic clustering based on protease gene expression. This result may be due to an interruption of normal interactions between RasIRaf signalling and transforming growth factor (TGF) P signalling, via Sma- and Mad- related protein (SMAD) signalling. These results demonstrate that the already identified link between mutations in kinase signalling, and aspects of gross colon tumour morphology (such as dysplasia) may be due to aberrant MMP expression patterning. The final aim of this research was to utilise methods developed in microdissection and specific Q-PCR analysis, to identify whether tumour-stroma differences in MMP gene expression might be used as markers of disease pathology. Total RNA from tumour, and biopsy-matched adjacent stromal tissue were segregated from 35 FFPE archival breast tumour biopsies. Comparison with stroma identified specific associations between TIMP-2 expression in the stroma and lymph node involvement, as well as stromelysin-3 (MMP-I I ) and TIMP-I expression and calcification of the tumour. Furthermore, a significant correlation was identified in the pattern of gelatinase (gelatinase AIMMP-2 & gelatinaseB1MMP-9) expression; while no significant correlation was identified in tumour-stroma MMP gene expression differences, and tumour grade, or hormone receptor status. These results suggest that coordinated changes within the tumour, and proximal stromal tissues (rather than tissue specific changes per se), regulate pathologically significant changes in breast carcinogenesis. In conclusion, this thesis describes the use of novel techniques in specific and global gene expression analysis that permitted examination of stromal gene expression changes in epithelial tumour progression. Microdissection facilitated localisation of expression to particular tissues, while cell culture models provided material with which to optimise and demonstrate the efficacy of techniques used (where tumour material itself was not abundant). Furthermore, we have identified significant and specific correlations between general stromal protease gene expression changes, a putative mammary epithelial differentiation factor (RANKL), alterations in growth factor signalling, and epithelial tumour pathology in the breast and colon. The combination of techniques developed in this study may assist in improvement of categorisation of tumours in clinical pathology. Specifically, the development of novel grading systems that link underlying molecular genetic changes with changes in tumour pathology. These processes may assist to improve diagnosis and provide more effective patient/tumour-specific drug therapies.

Plant alkaloids are secondary metabolites that may be synthesized in an inducible defense response to herbivory (Baldwin 1999). Genetic engineering of secondary metabolic pathways in plants to enhance or reduce metabolite production is limited by the current understanding of these pathways and their regulation in response to environmental conditions. This study was intended to provide new insights into the mechanism and regulation of alkaloid biosynthesis in N. tabacum by identifying genes that are coordinately regulated during conditions that induce alkaloid biosynthesis and by comparing their expression in regulatory mutant backgrounds that differ at two quantitative alkaloid loci, A and B. In order to identify novel genes that are differentially expressed during alkaloid biosynthesis, the transcriptional profiling procedure, fluorescent differential display (FDD), was used to screen total RNA isolated from Burley 21 (WT, AABB) and LA21 (low alkaloid regulatory mutant, aabb) tobacco root cultures that were induced for alkaloid synthesis. Four of thirteen cloned FDD fragments showed sequence homology to genes with defense-related functions. The differential expression of genes represented by selected FDD gene fragments was confirmed by comparing Northern blots of transcripts of those genes to known alkaloid biosynthetic genes, putrescine methyl transferase (PMT3), ornithine decarboxylase (ODC3), arginine decarboxylase (ADC1), and quinolinate phosphoribosyltransferase (QPRT). The role of the A and B loci in differential expression of genes represented by FDD clones and of known nicotine biosynthetic genes was examined using quantitative real time polymerase chain reaction (QRT-PCR) to measure transcript levels of these genes in four tobacco genotypes differing in alkaloid content, Burley 21(AABB), HI21 (AAbb), LI21(aaBB), and LA21 (aabb). Results of this study suggest that the A/B regulon is not limited to alkaloid biosynthetic genes, but includes multiple genes with defense-related functions. QRT-PCR analysis of nicotine biosynthetic genes and genes represented by confirmed differentially expressed FDD clones showed increased mRNA accumulation in response to alkaloid induction in all the tested genotypes, which suggests that the A and B mutations affect overall mRNA accumulation levels, rather than gene inducibility, per se. Baldwin, I.T. 1999. Inducible nicotine production in native Nicotiana as an example of adaptive phenotypic plasticity. Journal of Chem. Ecol. 25: 3-30.
Master of Science

Lee Wing Sum.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2000.
Includes bibliographical references (leaves 206-226).
Abstracts in English and Chinese.
Abstract --- p.i
Abstract (Chinese Version) --- p.iv
Acknowledgements --- p.vii
Table of Contents --- p.viii
List of Abbreviations --- p.xiv
List of Figures --- p.xvii
List of Tables --- p.xxiv
Chapter Chapter 1 --- Introduction --- p.1
Chapter Chapter 2 --- Literature review --- p.3
Chapter 2.1 --- Peroxisomes --- p.3
Chapter 2.2 --- Peroxisome proliferators --- p.5
Chapter 2.3 --- Human exposure pathways to peroxisome proliferators --- p.5
Chapter 2.4 --- Peroxisome proliferator-induced pleiotropic effects in rodents --- p.7
Chapter 2.4.1 --- Short-term effects --- p.7
Chapter 2.4.1.1 --- Hepatomegaly --- p.7
Chapter 2.4.2.1 --- Peroxisome proliferation --- p.8
Chapter 2.4.1.3 --- Alteration of gene transcriptions --- p.8
Chapter 2.4.2 --- Long-term effect --- p.9
Chapter 2.5 --- Mechanisms of actions of peroxisome proliferators --- p.9
Chapter 2.5.1 --- Substrate overload --- p.9
Chapter 2.5.2 --- Receptor-mediated --- p.11
Chapter 2.6 --- Peroxisome proliferator-activated receptors (PPARs) --- p.11
Chapter 2.6.1 --- Structure of PPARs --- p.11
Chapter 2.6.2 --- Tissue-specific expression of PPARs --- p.15
Chapter 2.6.3 --- Physiological functions of PPARs --- p.19
Chapter 2.6.3.1 --- PPARα --- p.19
Chapter 2.6.3.2 --- PPARγ --- p.21
Chapter 2.6.3.3 --- PPARδ --- p.23
Chapter 2.7 --- Role of PPARα involved in peroxisome proliferator-induced pleiotropic responses --- p.24
Chapter 2.7.1 --- Short-term effects --- p.24
Chapter 2.7.2 --- Long-term effect --- p.24
Chapter 2.8 --- Mechanisms of peroxisome proliferator-induced hepatocarcinogenesis --- p.25
Chapter 2.8.1 --- Oxidative stress --- p.25
Chapter 2.8.2 --- Suppression of apoptosis --- p.26
Chapter 2.8.3 --- Increased cell proliferation --- p.27
Chapter 2.9 --- Species difference to peroxisome proliferator-induced pleiotropic effects --- p.28
Chapter 2.10 --- Fluorescent differential display (FDD) --- p.32
Chapter Chapter 3 --- Objectives --- p.35
Chapter Chapter 4 --- Materials and methods --- p.37
Chapter 4.1 --- Animals and treatments --- p.37
Chapter 4.1.1 --- Materials --- p.37
Chapter 4.1.2 --- Methods --- p.37
Chapter 4.2 --- Serum triglyceride and cholesterol analyses --- p.39
Chapter 4.2.1 --- Materials --- p.41
Chapter 4.2.2 --- Methods --- p.41
Chapter 4.2.2.1 --- Serum preparation --- p.41
Chapter 4.2.2.2 --- Triglyceride determination --- p.41
Chapter 4.2.2.3 --- Cholesterol determination --- p.42
Chapter 4.3 --- Statistical analysis --- p.42
Chapter 4.4 --- Tail-genotyping --- p.42
Chapter 4.4.1 --- Materials --- p.44
Chapter 4.4.2 --- Methods. --- p.44
Chapter 4.4.2.1 --- Preparation of genomic tail DNA --- p.44
Chapter 4.4.2.2 --- PCR reaction --- p.45
Chapter 4.5 --- Total RNA isolation --- p.45
Chapter 4.5.1 --- Materials --- p.48
Chapter 4.5.2 --- Methods --- p.48
Chapter 4.6 --- DNase I treatment --- p.48
Chapter 4.6.1 --- Materials --- p.49
Chapter 4.6.2 --- Methods --- p.49
Chapter 4.7 --- Reverse transcription of mRNA and fluorescent PCR amplification --- p.50
Chapter 4.7.1 --- Materials --- p.50
Chapter 4.7.2 --- Methods --- p.53
Chapter 4.8 --- Fluorescent differential display (FDD) --- p.53
Chapter 4.8.1 --- Materials --- p.53
Chapter 4.8.2 --- Methods --- p.54
Chapter 4.9 --- Excision of differentially expressed cDNA fragments --- p.54
Chapter 4.9.1 --- Materials --- p.57
Chapter 4.9.2 --- Methods --- p.57
Chapter 4.10 --- Reamplification of differentially expressed fragments --- p.57
Chapter 4.10.1 --- Materials --- p.60
Chapter 4.10.2 --- Methods --- p.60
Chapter 4.11 --- Subcloning of reamplified cDNA fragments --- p.62
Chapter 4.11.1 --- PCR-TRAP® cloning system --- p.62
Chapter 4.11.1.1 --- Materials --- p.63
Chapter 4.11.1.2 --- Methods --- p.63
Chapter 4.11.2 --- AdvaTage´ёØ PCR cloning system --- p.65
Chapter 4.11.2.1 --- Materials --- p.65
Chapter 4.11.2.2 --- Methods --- p.66
Chapter 4.12 --- Purification of plasmid DNA from recombinant clones --- p.69
Chapter 4.12.1 --- Materials --- p.69
Chapter 4.12.2 --- Methods --- p.69
Chapter 4.13 --- DNA sequencing of differentially expressed cDNA fragments --- p.70
Chapter 4.13.1 --- CEQ 2000 Dye Terminator Cycle Sequence system --- p.71
Chapter 4.13.1.1 --- Materials --- p.71
Chapter 4.13.1.2 --- Methods --- p.71
Chapter 4.13.2 --- ABI PRISM´ёØ dRhodamine Terminator Cycle Sequencing system --- p.72
Chapter 4.13.2.1 --- Materials --- p.72
Chapter 4.13.2.2 --- Methods --- p.72
Chapter 4.13.3 --- Homology search against computer databases --- p.73
Chapter 4.14 --- Northern analysis of differentially expressed cDNA fragments --- p.73
Chapter 4.14.1 --- Formaldehyde gel electrophoresis of total RNA --- p.74
Chapter 4.14.1.1 --- Materials --- p.74
Chapter 4.14.1.2 --- Methods --- p.74
Chapter 4.14.2 --- Preparation of cDNA probes for hybridization --- p.74
Chapter 4.14.2.1 --- PCR DIG labeling --- p.75
Chapter 4.14.2.1.1 --- Materials --- p.75
Chapter 4.14.2.1.2 --- Methods --- p.75
Chapter 4.14.2.2 --- Random Prime cDNA DIG labeling --- p.75
Chapter 4.14.2.2.1 --- Materials --- p.75
Chapter 4.14.2.2.2 --- Methods --- p.76
Chapter 4.14.3 --- Purification of DNA from agarose gel --- p.77
Chapter 4.14.3.1 --- Materials --- p.77
Chapter 4.14.3.2 --- Methods --- p.78
Chapter 4.14.4 --- Hybridization --- p.78
Chapter 4.14.4.1 --- Materials --- p.78
Chapter 4.14.4.2 --- Methods --- p.73
Chapter 4.14.5 --- Synthesis of mouse GAPDH probe from normalization --- p.80
Chapter 4.14.5.1 --- Materials --- p.80
Chapter 4.14.5.2 --- Methods --- p.80
Chapter Chapter 5 --- Results --- p.82
Chapter 5.1 --- Liver morphology --- p.82
Chapter 5.2 --- Liver weight --- p.82
Chapter 5.3 --- Serum triglyceride and cholesterol levels --- p.88
Chapter 5.4 --- Confirmation of genotypes --- p.91
Chapter 5.5 --- DNase I treatment --- p.91
Chapter 5.6 --- FDD RT-PCR and band excision --- p.98
Chapter 5.7 --- Reamplification of excised cDNA fragments --- p.111
Chapter 5.8 --- Subcloning of reamplified cDNA fragments --- p.121
Chapter 5.9 --- DNA sequencing of subcloned cDNA fragments --- p.124
Chapter 5.10 --- Confirmation of the differentially expressed cDNA fragments by Northern blot analysis --- p.132
Chapter 5.11 --- Temporal expression pattern of differentially expressed genes --- p.157
Chapter 5.12 --- Tissue distribution pattern of differentially expressed genes --- p.171
Chapter Chapter 6 --- Discussions --- p.183
Chapter 6.1 --- "Lack of hepatomegaly, hypotriglyceridemia and hepatic nodule formation in PPARα (-/-) mice" --- p.184
Chapter 6.2 --- "Identification of PPARα-dependent and Wy-14,643 responsive genes" --- p.185
Chapter 6.3 --- Functional roles of the isolated cDNA fragments --- p.186
Chapter 6.3.1 --- Fragments B14 and H4 --- p.187
Chapter 6.3.2 --- Fragment H1 --- p.189
Chapter 6.3.3 --- Fragment H5 --- p.192
Chapter 6.3.4 --- Fragment H8 --- p.194
Chapter 6.4 --- Temporal expression patterns of the isolated cDNA fragments --- p.196
Chapter 6.5 --- Tissue distribution patterns of the isolated cDNA fragments --- p.197
Chapter Chapter 7 --- Conclusions --- p.200
Chapter Chapter 8 --- Future studies --- p.204
Chapter 8.1 --- Subcloning and characterization of the other differentially expressed genes --- p.204
Chapter 8.2 --- Overexpression and inhibition expression of specific genes --- p.204
Chapter 8.3 --- Generating transgenic mice with target disruption of specific gene --- p.205
References --- p.206

by Cheng Chi Wa.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1998.
Includes bibliographical references (leaves 110-120).
Abstract also in Chinese.
Title --- p.i
Abstract --- p.ii
Acknowledgement --- p.iv
Abbreviations --- p.v
Abbreviation Table for Amino Acids --- p.vi
Table of Contents --- p.vii
List of Figures --- p.xii
List of Tables --- p.xv
Chapter Chapter 1 --- Introduction --- p.1
Chapter Chapter 2 --- Literature review --- p.2
Chapter Part I --- Hypertrophic Scar
Chapter 2.1 --- Definition of hypertrophic scar --- p.2
Chapter 2.2 --- Pathology --- p.2
Chapter 2.3 --- Epidemiology findings --- p.3
Chapter 2.3.1 --- Ethnicity --- p.3
Chapter 2.3.2 --- Age --- p.3
Chapter 2.3.3 --- Body location --- p.3
Chapter 2.4 --- Mechanism of cutaneous wound healing --- p.4
Chapter 2.4.1 --- Phase I - Haemostasis and inflammation --- p.4
Chapter 2.4.1.1 --- Haemostasis --- p.6
Chapter 2.4.1.2 --- Early phase of inflammation --- p.6
Chapter 2.4.1.3 --- Late phase of inflammation --- p.7
Chapter 2.4.2 --- Phase II - Re-epithelialization --- p.7
Chapter 2.4.2.1 --- Migration of epidermal keratinocytes --- p.8
Chapter 2.4.2.2 --- Migration of fibroblasts --- p.8
Chapter 2.4.2.3 --- Angiogenesis --- p.9
Chapter 2.4.3 --- Phase III - Tissue remodeling --- p.10
Chapter 2.4.3.1 --- Cell maturation and apoptosis --- p.10
Chapter 2.4.3.2 --- Exrtracellular matrix remodeling --- p.10
Chapter 2.5 --- Alteration of wound healing - Possible pathogenic factors of hypertrophic scar --- p.11
Chapter 2.5.1 --- Changes in Phase I-Inflammation --- p.13
Chapter 2.5.2 --- Changes in Phase II - Re-epithelialization/ tissue formation --- p.14
Chapter 2.5.3 --- Changes in Phase III - Tissue remodeling --- p.15
Chapter 2.6 --- The Role of fibroblasts in the formation of hypertrophic scar --- p.16
Chapter 2.6.1 --- Functions of fibroblasts in wound healing --- p.16
Chapter 2.6.2 --- Suggested aetiological role in the formation of hypertrophic scar fibroblasts --- p.16
Chapter 2.6.2.1 --- Fibroproliferation disorder --- p.18
Chapter 2.6.2.2 --- Extracellular Matrix remodeling disorder --- p.18
Chapter a) --- CoUaqen --- p.18
Chapter b) --- Proteoglycan --- p.19
Chapter 2.6.2.3 --- Other differentially expressed factors --- p.20
Chapter 2.7 --- Treatment of hypertrophic scar --- p.21
Chapter Part II --- Differential Display
Chapter 2.8 --- Current approaches for the studies of differential gene expression --- p.23
Chapter 2.9 --- Comparison amongst different approaches --- p.23
Chapter 2.10 --- The strategy of Differential Display RT-PCR (DDRT-PCR) --- p.24
Chapter 2.11 --- The application of DDRT-PCR to identify differentially expressed genes --- p.26
Chapter Chapter 3 --- Aims and Strategies --- p.27
Chapter Chapter 4 --- Methods and Materials --- p.29
Chapter 4.1 --- Materials --- p.29
Chapter 4.2 --- Clinical specimen collection --- p.31
Chapter 4.3 --- Primary explant culture --- p.31
Chapter 4.4 --- Immunohistochemical staining --- p.32
Chapter 4.5 --- Total RNA extraction --- p.32
Chapter 4.6 --- DNase I digestion --- p.33
Chapter 4.7 --- Differential display-RTPCR (DD-RTPCR) --- p.33
Chapter 4.8 --- Polyacrylamide gel electrophoresis --- p.34
Chapter 4.9 --- Reamplification of the differentially expressed fragments --- p.35
Chapter 4.10 --- Molecular cloning of the DNA fragments --- p.35
Chapter 4.11 --- Screening and miniprep of the plasmid DNA --- p.36
Chapter 4.12 --- Cycle sequencing --- p.38
Chapter 4.13 --- Data analysis --- p.38
Chapter 4.14 --- RT-PCR --- p.39
Chapter 4.15 --- Probe labeling by PCR with DIG-dUTP --- p.40
Chapter 4.16 --- Southern blotting --- p.41
Chapter Chapter5 --- p.42
Chapter 5.1 --- Clinical Specimen --- p.42
Chapter 5.2 --- Primary explant culture --- p.42
Chapter 5.3 --- The total RNA extraction from the cultured fibroblast --- p.45
Chapter 5.4 --- Differential display RT-PCR --- p.47
Chapter 5.5 --- Reamplification of the DNA fragments --- p.49
Chapter 5.6 --- Molecular cloning of the DNA fragment --- p.53
Chapter 5.7 --- DNA sequencing of the inserts --- p.58
Chapter 5.8 --- Analysis and identification of the DNA sequences --- p.62
Chapter 5.9 --- Semi-quantitative RT-PCR analysis of the differentially expressed genes --- p.76
Chapter Chapter6 --- p.87
Chapter Part I --- Validity of the Findings
Chapter 6.1 --- The Limitation of Tissue Sampling --- p.87
Chapter 6.2 --- Tissue Culture model --- p.88
Chapter 6.3 --- Differential Display RT-PCR --- p.89
Chapter 6.3.1 --- Identification of the differentially expressed genes --- p.89
Chapter 6.3.2 --- Confirmation of the differentially expressed genes --- p.91
Chapter 6.4 --- Technical difficulties and Limitations --- p.92
Chapter 6.4.1 --- Sampling --- p.92
Chapter 6.4.2 --- Primary tissue culture --- p.93
Chapter Part II --- Significance and Future Studies
Chapter 6.5 --- Down-regulation of thrombospondin 1 (TSP 1) in the hypertrophic scar fibroblasts --- p.94
Chapter 6.6 --- Biochemical and biological functions of TSP1 --- p.96
Chapter 6.6.1 --- The biochemical functions of TSP1 --- p.96
Chapter 6.6.2 --- The biochemical functions of TSP1 --- p.97
Chapter 6.7 --- The role of TSP 1 in the pathogenesis of hypertrophic scar --- p.98
Chapter 6.7.1 --- Down-regulation of TSP 1 may be responsible for the excessive microvessels in hypertrophic scar --- p.98
Chapter 6.7.2 --- Down-regulation of TSP 1 may be responsible for the failure of the apoptosis of the fibroblasts in the hypertrophic scar --- p.101
Chapter 6.8 --- Expression of TSP 1 during wound healing --- p.103
Chapter 6.9 --- Expression of TSP 1 in hypertrophic scarring --- p.107
Chapter 6.10 --- Cytochrome b561 and its biological function --- p.109
Chapter 6.11 --- Future studies --- p.108
Chapter 6.11.1 --- The expression of TSP 1 in hypertrophic scarring and normal wound healing --- p.108
Chapter 6.11.2 --- The expression of cytochrome b561 --- p.109
Chapter 6.11.3 --- A full scale study of differential display RT-PCR --- p.109
References --- p.110
Appendices --- p.121
Chapter I --- The complete mRNA sequence of thrombospondin1 precursor --- p.121
Chapter II --- The mRNA sequence of cytochrome b561 --- p.123

At the beginning of this project, it was clear that R. albus adhered tightly to cellulose and its efficient degradation of this polysaccharide was dependent on micromolar concentrations of phenylacetic acid (PAA) and phenylpropionic acid (PPA). The objectives for our research were: i) to identify how many different kinds of cellulose binding proteins are produced by Ruminococcus albus; ii) to isolate and clone the genes encoding some of these proteins from the same bacterium; iii) to determine where these various proteins were located and; iv) quantify the relative importance of these proteins in affecting the rate and extent to which the bacterium becomes attached to cellulose. BARD support has facilitated a number of breakthroughs relevant to our fundamental understanding of the adhesion process. First, R. albus possesses multiple mechanisms for adhesion to cellulose. The P.I.'s laboratory has discovered a novel cellulose-binding protein (CbpC) that belongs to the Pil-protein family, and in particular, the type 4 fimbrial proteins. We have also obtained genetic and biochemical evidence demonstrating that, in addition to CbpC-mediated adhesion, R. albus also produces a cellulosome-like complex for adhesion. These breakthroughs resulted from the isolation (in Israel and the US) of spontaneously arising mutants of R. albus strains SY3 and 8, which were completely or partially defective in adhesion to cellulose, respectively. While the SY3 mutant strain was incapable of growth with cellulose as the sole carbon source, the strain 8 mutants showed varying abilities to degrade and grow with cellulose. Biochemical and gene cloning experiments have been used in Israel and the US, respectively, to identify what are believed to be key components of a cellulosome. This combination of cellulose adhesion mechanisms has not been identified previously in any bacterium. Second, differential display, reverse transcription polymerase chain reaction (DD RT-PCR) has been developed for use with R. albus. A major limitation to cellulose research has been the intractability of cellulolytic bacteria to genetic manipulation by techniques such as transposon mutagenesis and gene displacement. The P.I.'s successfully developed DD RT- PCR, which expanded the scope of our research beyond the original objectives of the project, and a subset of the transcripts conditionally expressed in response to PAA and PPA have been identified and characterized. Third, proteins immunochemically related to the CbpC protein of R. albus 8 are present in other R. albus strains and F. intestinalis, Western immunoblots have been used to examine additional strains of R. albus, as well as other cellulolytic bacteria of ruminant origin, for production of proteins immunochemically related to the CbpC protein. The results of these experiments showed that R. albus strains SY3, 7 and B199 all possess a protein of ~25 kDa which cross-reacts with polyclonal anti-CbpC antiserum. Several strains of Butyrivibrio fibrisolvens, Ruminococcus flavefaciens strains C- 94 and FD-1, and Fibrobacter succinogenes S85 produced no proteins that cross-react with the same antiserum. Surprisingly though, F. intestinalis strain DR7 does possess a protein(s) of relatively large molecular mass (~200 kDa) that was strongly cross-reactive with the anti- CbpC antiserum. Scientifically, our studies have helped expand the scope of our fundamental understanding of adhesion mechanisms in cellulose-degrading bacteria, and validated the use of RNA-based techniques to examine physiological responses in bacteria that are nor amenable to genetic manipulations. Because efficient fiber hydrolysis by many anaerobic bacteria requires both tight adhesion to substrate and a stable cellulosome, we believe our findings are also the first step in providing the resources needed to achieve our long-term goal of increasing fiber digestibility in animals.