Plant Molecular Biology Manual (Second Edition) is an entirely new manual containing both fundamental and recently described techniques in the area of plant molecular biology. Designed for use in the research laboratory, the Plant Molecular Biology Manual presents detailed techniques in the areas of plant transformation, recombinant DNA and other nucleic acid manipulations, nuclear run-on and in vitro transcription systems, in situ hybridization and immunodetection systems, protein-nucleic acid interaction analyses, subcellular targeting of proteins in the plant cell, and gene tagging using T-DNA and transposons. This second edition contains more than 40 newly written chapters, including descriptions of subjects such as virus-mediated gene transfer, specialized Agrobacterium strains and T-DNA vectors, nuclear run-on and in vitro transcription systems, non-radioactive detection systems, characterization of transcription factors, nuclear protein targeting, and T-DNA and transposon mutagenesis, not previously described in the first edition.

Plant Molecular Biology Manual

(1996). Progress with Proteome Projects: Why all Proteins Expressed by a Genome Should be Identified and How To Do It. Biotechnology and Genetic Engineering Reviews: Vol. 13, No. 1, pp. 19-50.

Progress with Proteome Projects: Why all Proteins Expressed by a Genome Should be Identified and How To Do It

Detection of differentially regulated genes has been severely hampered by technical limitations. In an effort to overcome these problems, the PCR-coupled subtractive process of representational difference analysis (RDA) [Lisitsyn, N. et al. (1993) Science 259, 946-951] has been adapted for use with cDNA. In a model system, RAG-1 and RAG-2, the genes responsible for activating V(D)J recombination, were identified in a genomic transfectant by cDNA RDA in a small fraction of the time taken by conventional means. The system was also modified to eliminate expected difference products to facilitate the identification of novel genes. Additional alterations to the conditions allowed isolation of differentially expressed fragments. Several caffeine up-regulated clones were obtained from the pre-B cell line 1-8, including IGF-1B, and a predicted homologue of the natural killer cell antigen, NKR-P1. The approach was found to be fast, extremely sensitive, reproducible, and predominantly lacked false positives. cDNA RDA has the capacity and adaptability to be applied to a wide range of biological problems, including the study of single gene disorders, characterization of mutant and complemented cell types, developmental or post-event expression time courses, and examination of pathogen-host interactions.

Identifying differences in mRNA expression by representational difference analysis of cDNA

involves alterations in specific patterns of gene expression. In order to screen for brain region specific mRNAs which are transcriptionally regulated by acute cocaine and amphetamine, PCR differential display was employed. This approach identified a previously uncharacterized mRNA whose relative levels in the striatum are induced four- to fivefold by acute psychomotor stimulant administration. Isolation and characterization of corresponding cDNA clones resulted in complete nucleotide sequence analysis, including prediction of the encoded protein product. Alternate polyA site utilization in the predicted 3′ noncoding region results in the appearance of an RNA doublet, approximately 700 and 900 bases in length, following Northern analysis. A presumed alternate splicing event further generates diversity within the transcripts, and results in the presence or absence of an in-frame 39 base insert within the putative protein coding region. As a result, the predicted translation products are either 129 or 116 amino acids in length. A common hydrophobic leader sequence at the amino terminus is present within each predicted polypeptide, suggesting that the protein product is targeted for entry into the secretory pathway. Basal expression of the RNA doublet is limited to neuroendocrine tissues, further implying that the protein product plays a functional role in both neuronal and endocrine tissues.

https://www.jneurosci.org/content/jneuro/15/3/2471.full.pdf

PCR differential display identifies a rat brain mRNA that is transcriptionally regulated by cocaine and amphetamine.

Differential display of mRNA was used to identify concordant changes in gene expression induced by two mood-stabilizing agents, lithium and valproate (VPA). Both treatments, on chronic administration, increased mRNA levels of the transcription factor polyomavirus enhancer-binding protein (PEBP) 2beta in frontal cortex (FCx). Both treatments also increased the DNA binding activity of PEBP2 alphabeta and robustly increased the levels of bcl-2 (known to be transcriptionally regulated by PEBP2) in FCx. Immunohistochemical studies revealed a marked increase in the number of bcl-2-immunoreactive cells in layers 2 and 3 of FCx. These novel findings represent the first report of medication-induced increases in CNS bcl-2 levels and may have implications not only for mood disorders, but also for long-term treatment of various neurodegenerative disorders.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1471-4159.1999.720879.x

The mood-stabilizing agents lithium and valproate robustly increase the levels of the neuroprotective protein bcl-2 in the CNS

We have significantly improved a method originally developed by Liang and Pardee [Science 257 (1992) 967-971] to display a broad spectrum of expressed genes and to detect differences in expression between different cell types. We have analysed various aspects of the technique and have modified it for both, the application to fast and efficient identification of genes and the use with automatic analysis systems. Based on the mathematical background we have devised the appropriate number of optimal PCR primers. We have also introduced nondenaturating gels for separating double stranded fragments as single bands. By applying the method to regenerating mouse liver, we have identified, out of a total of 38,000 bands, about 70 fragments where the expression of the corresponding genes seems to be differentially regulated at different time points. Application of the method to an automatic DNA sequencer was successfully done. Thus, we have confirmed the usefulness and increased the power of the RNA display technique, which we named differential display reverse transcription PCR (DDRT-PCR), and have extended the range of its application.

Identification of differentially expressed mRNA species by an improved display technique (DDRT-PCR).

Publisher Summary This chapter presents the analysis of altered gene expression by differential display. Temporal and spatial expression of the 100,000 different genes in a mammalian cell I must be highly regulated and tightly controlled as specificity determines life processes from the fertilized egg to death of the organism. The course of normal development as well as the pathological changes that arise in diseases such as cancer, whether caused by a single gene mutation or complex multigene effects, are driven by changes in gene expression. A fingerprinting technique for mRNAs would be very useful, for instance, by 2D electrophoresis, similar to 2D protein separation. A novel method named "differential display" (DDRT-PCR), according to polymerase chain reaction (PCR) terminology, was developed. This method provides a sensitive and flexible approach to the identification of genes that are differentially expressed at the mRNA level. The method is directed toward the identification of differentially expressed genes, detecting individual mRNA species that are changed in different sets of eukaryotic cells, and then permitting recovery and cloning of their DNA.

Analysis of altered gene expression by differential display.

Agent for diagnosing gene expression comprises at least 288 oligonucleotide primer pairs formed from a pool of at least 24 5'-oligonucleotide primers and at least 12 3'-oligonucleotide primers coupled with a label which is recognised in a detection procedure. Also claimed is a method for using the agent comprising (a) transcribing total cell RNA into cDNA; (b) amplifying the cDNA with the agent in a specified number, pref. 288-360, of polymerase chain reactions (PCR's); (c) sepg. each of the resulting fragment mixts. using a non-denatured polyacrylamide gel; (d) registering the band pattern and comparing it with a standard; and (e) opt. isolating the DNA from the bands and using it as a probe for the gene isolation. USE/ADVANTAGE - The agent and method are used in clinical diagnoses, e.g. of cancer, and in molecular biology to identify and isolate expressed genes. Gene identification can be performed rapidly, automatically and more precisely than in known procedures, e.g. Science 257, 1992, 967 and Cancer Research 52, 1992, 6966.

Diagnostic agent for investigating gene expression - comprises oligonucleotide primer pairs formed from labelled 5'- and 3'-oligonucleotide primers

A complex diagnostic agent of genetic expression consists of a set of at least 288 oligonucleotide primer pairs composed of a reserve of at least 24 5'-oligonucleotide primers and at least 12 3'-oligonucleotide primers. The length of the oligonucleotide primers lies between 6 and 13 nucleotides, and preferably equals 10 nucleotides. The sequences of the 5'-oligonucleotide primers preferably have the same number of G+C and A+T. With this agent, at least 288 parallel and independent PCR tests are carried out in a cDNA mixture. After separation in non-denaturating polyacrylamide gels, the line pattern is recorded and compared with the standard of a reference cell. The fragment profiles are preferably automatically recorded by colour marked primers. The invention has applications in molecular biology, in particular for identifying known and unknown expressed genes and for isolating genes, as well as in clinical diagnosis, for example for detecting morbid changes, including cancer.

David Bauer

Papers

Identification of differentially expressed mRNA species by an improved display technique (DDRT-PCR).

Analysis of altered gene expression by differential display.

Diagnostic agent for investigating gene expression - comprises oligonucleotide primer pairs formed from labelled 5'- and 3'-oligonucleotide primers

Complex diagnostic agent of genetic expression and medical diagnosis and gene isolation process using said diagnostic agent