Thesis (MSc)- Stellenbosch University, 2001. / ENGLISH ABSTRACT: Wood is composed of three main components: cellulose, hemicellulose and lignin.
Cellulose is the main structural polymer, whereas the function of lignin in plants is to
impart rigidity to the cells, to waterproof the vascular system, and to protect the plant
against pathogens. A group of microorganisms, called white-rot fungi, are able to
selectively degrade the lignin and hemicellulose from wood leaving the cellulose virtually
untouched. The most widely studied fungus of this group is the basidiomycete
Phanerochaete chrysosporium, which has become a model organism in studies of lignin
degradation.
Lignin is a large, heterogenous and water insoluble polymer and therefore the enzymes
needed to degrade it have to be extracellular and non-specific. There are a number of
enzymes that are involved in the degradation of lignin, including lignin peroxidases,
manganese dependent peroxidases and laccases. Laecases are blue copper oxidases that
require molecular oxygen to function, whereas lignin peroxidases and manganese
peroxidases are heme proteins that require hydrogen peroxide. Phanerochaete
chrysosporium has all three of these enzymes, as well as a system for producing the
hydrogen peroxide that is necessary for peroxidases to function.
For both scientific and industrial purposes, it is important to obtain linkage maps of the
positions of genes in the genome of an organism. Most fungi, including P. chrysosporium,
lack easily identifiable phenotypical markers that can be used to map the position of genes
relative to each other on the genome. Previous methods of mapping genes in
P. chrysosporium involved auxotrophic mutants, radioactivity, or the use of hazardous
chemicals. Here we describe an automated DNA-sequencing based mapping technique
that eliminates many of the problems associated with previous techniques. Portions of the
genes to be mapped were amplified from homokaryotic single basidiospore cultures using
gene specific primers using the polymerase chain reaction (PCR) technique. The PCR
products were sequenced to determine the segregation of alleles. Two previously mapped
lignin peroxidases, lipA and lipC, were used to develop this method, and the results
obtained corresponded to the known genetic linkage. A newly characterised 13-glucosidase
encoding gene from P. chrysosporium was also mapped. Linkage was found between the
13-glucosidase gene and a histone (Hl) encoding gene.
In P. chrysosporium the lignin peroxidase isozymes are encoded by a family of at least ten
genes. Previous studies with P. chrysosporium BKM-F-1767 in defined media, wood and
soil have shown differential expression of the lignin peroxidase isozymes. In this
investigation the levels of expression of lignin peroxidases in P. chrysosporium ME446
cultures grown in nitrogen or carbon limited defined liquid media, as well as on aspen
wood chips were determined by competitive reverse transcriptase polymerase chain
reaction (RT-peR). These results were compared to those previously obtained from
P. chrysosporium BKM-F-1767 to evaluate strain specific variation in the expression of
lignin peroxidases. The results indicate that, although there were many similarities in the
patterns of lignin peroxidase expression, there were also enough differences to conclude
that there were strain specific variations in the temporal expression of the lignin
peroxidases.
To conclude, a fast and cost effective method for mapping genes in P. chrysosporium was
developed. Also, we showed that strain specific variation in temporal expression of lignin
peroxidases occurs. / AFRIKAANSE OPSOMMING: Hout bestaan uit drie hoof komponente nl. sellulose, hemisellulose en lignien. Sellulose is
die hoof strukturele polimeer, terwyl die funksie van lignin in plante is om die selle te
versterk, die vaskulêre sisteem waterdig te hou, en die plant teen patogene te beskerm. 'n
Groep mikroërganisms, bekend as witvrotswamme, kan lignien en hemisellulose selektief
uit die hout verwyder, terwyl die sellulosevesels oorbly. Vanuit hierdie groep swamme is
die meeste navorsing op die basidiomiseet Phanerochaete chrysosporium gedoen
Lignien is 'n groot, heterogene polimeer en is onoplosbaar in water. Die ensieme wat
benodig word om lignien afte breek is daarom nie-spesifiek en kom ekstrasellulêr voor. 'n
Aantal ensieme is by die afbraak van lignien betrokke, insluitend lignienperoksidase,
mangaanperoksidase en lakkase. Lakkase is 'n blou koperoksidase wat suurstof benodig
vir aktiwiteit. Lignienperoksidase en mangaanperoxidase is heemproteïene en benodig
waterstofperoksied. Phanerochaete chrysosporium het al drie van hiedie ensieme, sowel
as 'n sisteem wat waterstofperoksied produseer.
Vir beide wetenskaplike en nywerheidsdoeleindes is koppelingskaarte wat die posisie van
gene in die genoom van 'n organisme aandui noodsaaklik. Die meeste swamme,
P. chrysosporium ingesluit, het geen fenotipiese merkers wat maklik van mekaar onderskei
kan word nie, en dit is dus moeilik om 'n kaart van die ligging van gene op die genoom te
bepaal. Vorige metodes om gene in P. chrysosporium te karteer het auksotrofiese mutante,
radioaktiwiteit of gevaarlike chemikalieë gebruik. Ons beskryf 'n metode wat van
automatiese DNA-volgordebepaling gebruik maak en wat baie van die tekortkominge van
die ou metodes oorkom. Dele van die gene is met geen-spesifieke PKR-amplifikasie uit
kulture van homokariotiese enkel basidiospore verkry en die DNA-volgorde is bepaal om
die segregasie van die allele te ondersoek. Twee gene waarvoor 'n koppelingskaart alreeds
uitgewerk is, fipA en lipt), was gebruik om hierdie metode te ontwikkel. Die resultate stem
ooreen met die bekende genetiese koppeling tussen hierdie gene. 'n Geen wat onlangs in
P. chrysosporium ontdek is, nl. I3-glucosidase, is ook met hierdie metode gekarteer.
Koppeling is met 'n histoon (Hl) geen gevind.
Die lignienperoksidase isoensieme in P. chrysosporium word deur 'n familie van ten
minste tien gene gekodeer. Vorige navorsing met P. chrysosporium BKM-F-1767 in
gedefineerde media, hout en grond het getoon dat 'n variasie in die uitdrukking van lignienperoxidase isoensieme voorkom. In hierdie ondersoek is 'n kultuur van
P. chrysosporium ME446 in stikstof- of koolstof-beperkende vloeibare media opgegroei,
as ook op aspen houtblokkies. Die vlak van uitdrukking van die lignienperoksidases is deur
middel van die omgekeerde transkripsie polimerasekettingreaksie (RT-PKR) bepaal. Die
resultate vir P. chrysosporium ME446 is vergelyk met vorige resultate van
P. chrysosporium BKM-F-1767 om te bepaal of stamspesifieke variasies in die uitdrukking
van lignienperoksidases voorkom. Daar is 'n aanduiding dat, alhoewel soortgelyke patrone
in die vlakke van lignienperoksidase uitdrukking voorkom, daar ook noemenswaardige
verskille is. Hieruit kan afgelui word dat stamverwante variasie van lignienperokisdase
uitdrukking voorkom.
Ten slotte, ons het 'n vinnige, goedkoop metode om die gene in P. chrysosporium te
karteer ontwikkel. Ons het ook bewys dat stam-spesifieke variasie in die uitdrukking van
die lignienperoxidase gene voorkom.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52323 |
| Date | 12 1900 |
| Creators | Allsop, Simon |
| Contributors | Janse, B. J. H., Van Zyl, W. H., De Koker, T. H., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 82 p. : ill |
| Rights | Stellenbosch University |
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