Use of Complex DNA and Antibody Microarrays as Tools in Functional Analyses

While the deciphering of basic sequence information on a genomic scale is yielding complete genomic sequences in ever-shorter intervals, experimental procedures for elucidating the cellular effects and consequences of the DNA-encoded information become critical for further analyses. In recent years, DNA microarray technology has emerged as a prime candidate for the performance of many such functional assays. Technically, array technology has come a long way since its conception some 15 years ago, initially designed as a means for large-scale mapping and sequencing

Physical mapping and gene expression analysis in the genome of Neurospora crassa

The filamentous fungus Neurospora crassa has been used as a model organism in basic science for more than 50 years. In germany, a neurospora genome project started in 1998 that aims at the sequencing of the chromosomes II and V.The physical maps described in the first part of this work formed the basis of the sequencing project. For mapping, a cosmid library with a 17-fold genomic coverage and a BAC library with a 15-fold coverage were used, from which chromosome specific sublibraries were selected. Physical mapping was done by hybridising labelled DNA of single clones to the libraries. Ordering of the clones according to their hybridisation pattern was performed with an established software package. The resulting map of chromosome II contains 13 regions, the map of chromosome V 21 regions of overlapping clones (contigs).In the second part of the work some gene expression profiling experiments were performed using selfmade cDNA microarrays of N. crassa. By hybridising complex RNA probes transcriptional profiles were generated for three different growth conditions of mycelium of N. crassa. In each experiment, two mRNA populations were labelled by reverse transkription with two different fluorescent dyes and cohybridised to a single chip. After detection and quantification of the signal intensities, factors for induction or repression of the genes were calculated and visualised in a correspondence analysis

What's in the genome of a ®lamentous fungus? Analysis of the Neurospora genome sequence

The German Neurospora Genome Project has assembled sequences from ordered cosmid and BAC clones of linkage groups II and V of the genome of Neurospora crassa in 13 and 12 contigs, respectively. Including additional sequences located on other linkage groups a total of 12 Mb were subjected to a manual gene extraction and annotation process. The genome comprises a small number of repetitive elements, a low degree of segmental duplications and very few paralogous genes. The analysis of the 3218 identi®ed open reading frames provides a ®rst overview of the protein equipment of a ®lamentous fungus. Signi®cantly, N.crassa possesses a large variety of metabolic enzymes including a substantial number of enzymes involved in the degradation of complex substrates as well as secondary metabolism. While several of these enzymes are speci®c for ®lamentous fungi many are shared exclusively with prokaryotes