Both exon 1 and intron 1 of the maize Shrunken-1 (Sh1) gene individually stimulate expression of reporter genes in transient gene expression experiments if present within the transcription unit. The Sh1 exon 1 mediates a 10-fold increase in activity when inserted at the 5' end of the bacterial chloramphenicol transacetylase (CAT) marker gene in both monocot and dicot protoplasts. The Sh1 intron 1 enhances chimeric gene expression in rice and maize protoplasts approximately 100-fold but inhibits CAT expression in tobacco protoplasts. In combination, the stimulatory effects of Sh1 exon 1 and intron 1 are multiplicative in monocot protoplasts resulting in a final enhancement of up to 1000-fold compared to the unmodified CAT or luciferase marker genes.

The Glossy2 (Gl2) locus of maize is required for the formation of the epicuticular wax layer of young plants. gl2 mutant seedlings can be visually identified because of their glossy leaf surface which is different from the dull surface of wild-type seedlings. The Gl2 locus was isolated by transposon tagging. Seven unstable mutations, gl2-m2 to gl2-m8, were induced in a parental strain carrying an active transposable Activator (Ac) element in the unstable wx-m7 allele. Genetic tests on the gl2-m2 allele indicated that it was not caused by the Ac element but by the insertion of the transposable element Enhancer/Suppressor-Mutator (En/Spm). A Sa/l restriction fragment segregating with the mutant phenotype was identified, by Southern analysis, using sequences from the En/Spm element as a probe. Part of the fragment was cloned and was shown to carry part of the unstable gl2-m2 allele. These gl2 sequences were used to identify a genomic fragment carrying the wild-type allele and to isolate its corresponding cDNA sequence. The predicted Glossy2 protein consists of 426 amino acids. No similar amino acid sequence was found in protein data banks and the biochemical function of the Gl2 gene product is still unknown. The wild-type Gl2 transcript is found predominantly in juvenile leaves. The transcript level in the leaves of seedlings homozygous for a stable recessive gl2-ref allele is hardly detectable.

Background: Phi29 polymerase based amplification methods provides amplified DNA with minimal changes in sequence and relative abundance for many biomedical applications. RNA virus detection using microarrays, however, can present a challenge because phi29 DNA polymerase cannot amplify RNA nor small cDNA fragments (<2000 bases) obtained by reverse transcription of certain viral RNA genomes. Therefore, ligation of cDNA fragments is necessary prior phi29 polymerase based amplification. We adapted the QuantiTect Whole Transcriptome Kit (Qiagen) to our purposes and designated the method as Whole Transcriptome Amplification (WTA).

Single cell genome analysis methods are powerful tools to define features of single cells and to identify differences between them. Since the DNA amount of a single cell is very limited, cellular DNA usually needs to be amplified by whole-genome amplification before being subjected to further analysis. A single nucleus only contains two haploid genomes. Thus, any DNA damage that prevents amplification results in loss of damaged DNA sites and induces an amplification bias. Therefore, the assessment of single cell DNA quality is urgently required. As of today, there is no simple method to determine the quality of a single cell DNA in a manner that will still retain the entire cellular DNA for amplification and downstream analysis. Here, we describe a method for whole-genome amplification with simultaneous quality control of single cell DNA by using a competitive spike-in DNA template.

Düsseldorf Injury of peripheral nerve in mammals leads to a complex but stereotypical pattern of histological events that comprise a highly reproducible sequence of degenerative reactions (Wallerian degeneration) succeeded by regenerative responses. These reactions are based on a corresponding sequence of cellular and molecular interactions that, in turn, reflect the differential expression of specific genes with functions in nerve degeneration and repair. We report on more than 60 genes and their products that show a specific pattern of regulation following peripheral nerve lesion. The group of regulated genes encoding, e.g., transcription factors, growth factors and their receptors, cytokines, neuropeptides, myelin proteins and lipid carriers, and cytoskeletal proteins as well as extracellular matrix and cell adhesion molecules. We describe and compare the distinct time-courses and cellular origin of expression and further discuss established or putative molecular interrelationships and functions with respect to the contribution of these genes/gene products to the molecular regeneration program of the PNS. NEUROSCIENTIST 3:112-122, 1997