The "ups" and "downs" in Using Subtractive Cloning Techniques to Isolate Regulated Genes in Fish

Over the last decade, subtractive cloning approaches have beenused extensively to isolate genes that are up- or down-regulatedunder various conditions. These techniques have provided thefoundation for many subsequent studies concerning gene functionand regulation and, as such, have been valuable tools for manybiological fields. Over the past 10 years, we have used differentsubtractive cloning approaches to isolate genes in fish thatare regulated in relation to hormonal stimulation or the stageof ovarian maturation. These include conventional cDNA subtractionfollowed by library screening, differential display PCR, suppressionsubtraction hybridization, and more recently, iterative PCRsubtraction. We continue to use these techniques for the isolationof new genes involved in physiological processes in fish andbivalve molluscs. Examples that illustrate the use of thesedifferent subtractive cloning techniques are described, includingwhere possible the advantages and disadvantages of each. Inaddition, the use of ancillary methods (e.g., "Reverse Northerns")to facilitate the use of these subtractive approaches are discussed.     

Cloning Streptomyces genes for antibiotic production

The cloning and recombination of the genes of Streptomyces bacteria offer a method of increasing antibiotic yields and generating new antibiotics. Novel vectors, both plasmids and phages, have been developed for use with Streptomyces. This article describes some of these vectors and relevant cloning and screening techniques.     

Improved Screening of cDNAs Generated by mRNA Differential Display Enables the Selection of True Positives and the Isolation of Weakly Expressed Messages

The high percentage of false positives generated by differential display (as high as 85%) has previously limited the potential of the method. This report describes an efficient methodology that enables false positives to be discarded prior to cloning, via reverse Northern analysis. This first step of the screening also allows the detection of putative low abundance differential clones. Following cloning, a second reverse Northern combined with partial DNA sequencing and RT-PCR detection allows isolation of all differential cDNAs including very low abundance clones. Use of the sequential screening procedure described here led to the isolation of novel tomato genes responding to the plant hormone ethylene while minimising labor and materials input.     

Simple and rapid screening method for microbial D-stereospecific peptidase and esterase

The use of a turbid plate containing Z-D-Ala-D-AlaOBzl was a convenient method for the screening of microorganisms with D-stereospecific peptidase activity as well as D-stereospecific esterase activity. This paper discusses the suitability of the developed method and its application to molecular cloning of D-stereospecifc peptidase and esterase genes.     

Amplification of transcription of separate mitochondrial genes in human and monkey B-cell non-Hodgkin's lymphoma

Mitochondrial genes that are overexpressed in human and monkey B-cell non-Hodgkin lymphomas (B-NHLs) were sought via subtraction hybridization, cloning, and differential screening of the resulting cDNA libraries. The cDNAs of mitochondrial genes made an appreciable proportion of all lymphoma-specific cDNAs. Lymphomogenesis was associated with overexpression of a mitochondrial gene set which varied with lymphoma type and always included NADHIV. A possible association between overexpression of certain mitochondrial genes and cell malignant transformation is discussed.     

Increased Expression of Several Mitochondrial Genes in Human and Monkey B-Cell Non-Hodgkin Lymphomas

Mitochondrial genes overexpressed in human and monkey B-cell non-Hodgkin lymphomas (B-NHLs) were sought via subtraction hybridization, cloning, and differential screening of the resulting cDNA libraries. The cDNAs of mitochondrial genes constituted an appreciable proportion of all lymphoma-specific cDNAs. Lymphomogenesis was associated with upregulation of a set of mitochondrial genes, which varied with lymphoma type but always included NADHIV. A possible association between upregulation of certain mitochondrial genes and cell malignant transformation is discussed.     

A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in <Emphasis Type="Italic">E. coli</Emphasis>

Background  

The selection of bacterial recombinants that harbour a desired insert, has been a key factor in molecular cloning and a series of screening procedures need to be performed for selection of clones carrying the genes of interest. The conventional cloning techniques are reported to have problems such as screening high number of colonies, generation of false positives, setting up of control ligation mix with vector alone etc.     

YAC克隆的筛选及鉴定分析技术

人工酵母染色体（ＹＡＣ）技术是人类基因组分析及疾病相关基因的分离、克隆中的关键技术。在基因组ＹＡＣ文库基础上特定目的基因的分离克隆涉及ＹＡＣ克隆的筛选,嵌合体、缺失体和共转染克隆的检测与处理,插入片段的分离及其结构特征的分析,亚克隆的快速构建等等。近年来,有关技术取得了重要进展,已趋于成熟,并正得到广泛应用 。     

Modified differential display technique that eliminates radioactivity and decreases screening time

Several techniques are available that detect variations in gene expression between cellular populations. These include subtractive hybridization (SH), differential colony hybridization (DCH) and mRNA differential display, all based on the analysis of mRNA. The first two techniques, however, are limited because they require large amounts of mRNA for SH or several rounds of screening for DCH. Differential display overcomes both of these limitations. However, the conventional differential display technique is plagued by false positives and is labor intensive. The identification of genes that are truly differentially expressed, therefore, becomes a formidable task. We describe a modified differential display technique that overcomes the limitations of the conventional technique. This new technique eliminates a source of false positives, decreases the time required to screen a set of primers and reduces the use of radioactivity.     

Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes

Recent awareness that most microorganisms in the environment are resistant to cultivation has prompted scientists to directly clone useful genes from environmental metagenomes. Two screening methods are currently available for the metagenome approach, namely, nucleotide sequence-based screening and enzyme activity-based screening. Here we have introduced and optimized a third option for the isolation of novel catabolic operons, that is, substrate-induced gene expression screening (SIGEX). This method is based on the knowledge that catabolic-gene expression is generally induced by relevant substrates and, in many cases, controlled by regulatory elements situated proximate to catabolic genes. For SIGEX to be high throughput, we constructed an operon-trap gfp-expression vector available for shotgun cloning that allows for the selection of positive clones in liquid cultures by fluorescence-activated cell sorting. The utility of SIGEX was demonstrated by the cloning of aromatic hydrocarbon-induced genes from a groundwater metagenome library and subsequent genome-informatics analysis.     

基于PCR的基因差异表达分析技术

基因差异表达分析是研究许多生物学过程的分子基础的一条直接、有效的途径。自DDRT-PCR技术建立以来,一系列基于PCR的基因差异表达分析技术,如SAGE、SSH、RDA和DNA微阵列等相继发展起来,为分析和克隆差异表达的基因提供了更为快速、灵敏的工具。本对这几种方法进行了简要综述,比较了不同方法的优缺点,并展望了今后基因差异表达研究技术的发展方向。     

Improved assembly of multimeric genes for the biosynthetic production of protein polymers

We report a general method for the construction of highly repetitive synthetic genes and their use in the biosynthetic production of artificial protein polymers. Through the application of improved recombinant DNA techniques and high-throughput screening methods, we have developed a facile approach to rapid gene assembly and cloning which is widely applicable in the biosynthesis of novel protein polymers. Using this technique, synthetic genes encoding tandem repeats of the beta-sheet forming amino acid sequence AEAEAKAK were constructed and subsequently cloned into a bacterial expression host for inducible protein production. A 17-kDa fusion protein, poly-EAK9, was isolated from Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. The amino acid sequence and molecular weight were confirmed by amino acid analysis, N-terminal sequencing, and MALDI-TOF mass spectrometry. Circular dichroism studies on the artificial protein poly-EAK9 demonstrate the formation of a beta-sheet structure in aqueous solution.     

Bacteriophage lambda-based expression vectors

Bacteriophage lambda has been in use as a cloning vector for over 25 years, and has been used extensively as an expression vector. The efficiency of packaging and infection, and the simplicity of plaque screening are advantages of lambda as a cloning vector. A number of ingenious modifications help overcome the disadvantages associated with its mode of growth and its size. Some lambda vectors have been designed to be readily converted into plasmids or phagemids, and there are a variety of promoters and fusions that can be used to drive expression of foreign genes. Screening lambda libraries with antibodies or ligands is a powerful way of identifying novel genes.     

Application and limitations of differential hybridization in the isolation of T cell-specific cDNA clones

We investigated the limitations and effectiveness of differential hybridization in the cloning of T cell-specific cDNA (complementary DNA) molecular clones. By using the technique with T cell and B cell cDNA probes, together with Northern blot analysis, we successfully isolated cDNA clones exclusively expressed in T cells from 1 X 10(4) plaque-forming units of a T cell hybridoma. These clones represent 0.068% of the mass of the cytoplasmic mRNA. Our result shows that differential hybridization is an effective procedure when used in combination with Northern blot analysis for screening of genes selectively expressed in T cells.     

The activation of cellular genes in transformed cells

We have used differential cDNA cloning techniques to isolate a number of genes that are activated as a result of transformation by the DNA tumour virus Simian virus 40. From the nucleotide sequences of the cDNA clones we have been able to identify some of these genes. One of them derives from the major histocompatibility complex and contains a repetitive element that identifies a large number of RNAs present in pluripotential embryonic cells.     

Flow cytometry-assisted cloning of specific sequence motifs from complex 16S rRNA gene libraries

A flow cytometry method was developed for rapid screening and recovery of cloned DNA containing common sequence motifs. This approach, termed fluorescence-activated cell sorting-assisted cloning, was used to recover sequences affiliated with a unique lineage within the Bacteroidetes not abundant in a clone library of environmental 16S rRNA genes.     

Flow Cytometry-Assisted Cloning of Specific Sequence Motifs from Complex 16S rRNA Gene Libraries

A flow cytometry method was developed for rapid screening and recovery of cloned DNA containing common sequence motifs. This approach, termed fluorescence-activated cell sorting-assisted cloning, was used to recover sequences affiliated with a unique lineage within the Bacteroidetes not abundant in a clone library of environmental 16S rRNA genes.     

Targeted gene walking polymerase chain reaction.

We describe a modification of a polymerase chain reaction method called 'targeted gene walking' that can be used for the amplification of unknown DNA sequences adjacent to a short stretch of known sequence by using the combination of a single, targeted sequence specific PCR primer with a second, nonspecific 'walking' primer. This technique can replace conventional cloning and screening methods with a single step PCR protocol to greatly expedite the isolation of sequences either upstream or downstream from a known sequence. A number of potential applications are discussed, including its utility as an alternative to cloning and screening for new genes or cDNAs, as a method for searching for polymorphic sites, restriction endonuclease or regulatory regions, and its adaptation to rapidly sequence DNA of lengthy unknown regions that are contiguous to known genes.