47个早期人胚胎低丰度表达基因ESTs筛选及结果分析

构建高质量ｃＤＮＡ文库在基因克隆、ｍＲＮＡ差异展示、表达序列标签测序和基因定位等研究中具有十分重要的作用。为了从早期胚胎中分离人类新基因,构建了受精后３周龄的人ｃＤＮＡ文库,用标记的一链ｃＤＮＡ探针对该文库的６５０８个克隆子进行菌落原位杂交,得到１６７７个无任何杂交信号的低丰度表达克隆子,从中随机挑选了４７个进行５′端部分测序,将测序结果与三大基因库进行序列同源性比较,发现１８个克隆（３８．３％）     

Bovine elastin cDNA clones: Evidence for the occurrence of a new elastin-related protein in fetal calf ligamentum nuchae

Poly (A+) mRNA was isolated from fetal calf ligamenturn nuchae and used for the construction of cDNA libraries. A fraction highly enriched in elastin mRNA was used to prepare the cDNA probes for screening the libraries. A 2 kb clone, pREl, gave the most positive signal in colony hybridization. It hybridized to a mRNA of the same size as reported for elastin mRNAs from chick and sheep. Hybrid-arrested translation showed that translation of mRNAs for proteins other than elastin doublet was not inhibited by pREI. Southern blot analysis showed that pREl has sequence homology with pVE6 and pVE10, which were tentatively identified as elastin-related cDNA clones representing two distinct mRNAs. DNA sequence data from the 5 end of pREl show that the translated amino acid sequence is not typical of known elastin sequences but contains some elastin-like sequences. All of this evidence strongly suggests the occurrence in fetal calf nuchal ligament of a mRNA which codes for a previously unknown elastin-related protein.     

Subtraction hybridization cDNA libraries from colon carcinoma and hepatic cancer

cDNA clones of differentially expressed mRNAs in a colon carcinoma and a hepatocellular carcinoma have been isolated by subtractive cDNA cloning. The subtracted material is at least 90 X enriched for differentially expressed sequences and can be used for construction of subtractive cDNA libraries and polymerase chain reaction (PCR) amplification to generate differential probes. Commercially available lambda ZAP II is used for construction of primary libraries since single-stranded phage bearing the cloned cDNA can be excised in vivo and because lambda libraries are convenient for subsequent screening and manipulations. Rare mRNAs (less than 0.01% abundance), which are differentially expressed, can be isolated utilizing this procedure.     

Localization of types I, II, and III collagen mRNAs in developing human skeletal tissues by in situ hybridization

Paraffin sections of human skeletal tissues were studied in order to identify cells responsible for production of types I, II, and III collagens by in situ hybridization. Northern hybridization and sequence information were used to select restriction fragments of cDNA clones for the corresponding mRNAs to obtain probes with a minimum of cross-hybridization. The specificity of the probes was proven in hybridizations to sections of developing fingers: osteoblasts and chondrocytes, known to produce only one type of fibrillar collagen each (I and II, respectively) were only recognized by the corresponding cDNA probes. Smooth connective tissues exhibited variable hybridization intensities with types I and III collagen cDNA probes. The technique was used to localize the activity of type II collagen production in the different zones of cartilage during the growth of long bones. Visual inspection and grain counting revealed the highest levels of pro alpha 1(II) collagen mRNAs in chondrocytes of the lower proliferative and upper hypertrophic zones of the growth plate cartilage. This finding was confirmed by Northern blotting of RNAs isolated from epiphyseal (resting) cartilage and from growth zone cartilage. Analysis of the osseochondral junction revealed virtually no overlap between hybridization patterns obtained with probes specific for type I and type II collagen mRNAs. Only a fraction of the chondrocytes in the degenerative zone were recognized by the pro alpha 1(II) collagen cDNA probe, and none by the type I collagen cDNA probe. In the mineralizing zone virtually all cells were recognized by the type I collagen cDNA probe, but only very few scattered cells appeared to contain type II collagen mRNA. These data indicate that in situ hybridization is a valuable tool for identification of connective tissue cells which are actively producing different types of collagens at the various stages of development, differentiation, and growth.     

A family of lambda phage cDNA cloning vectors, lambda SWAJ, allowing the amplification of RNA sequences

This paper describes the construction and characterization of a family of lambda phage cDNA cloning vectors that allows high-efficiency directional cDNA cloning and selective amplification of either sense or antisense cRNA sequences. These vectors contain several unique restriction sites (EcoRI, XbaI, and SacI) positioned between two specific phage promoters, SP6 and T7. This system facilitates the in vitro preparation of single-stranded (ss) RNA molecules that should be useful in subtractive hybridization and in situ hybridization procedures. Using subtractive hybridization and this vector system, it should be possible to identify sequences present in one cDNA library and not another. In addition, it should be possible to construct subtracted cDNA libraries in these vectors and to generate high specific activity, ss, antisense cRNA probes directly from DNA prepared from the whole subtracted library or from individual clones.     

In situ localization with digoxigenin-labelled probes of tau-related mRNAs in the rat pancreas

Summary Two cDNA probes complementary to fetal rat brain tau cDNA were produced by the polymerase chain reaction (PCR) and labelled by digoxigenin-11-dUTP incorporation during the PCR elongation step. These probes were tested for thein situ localization of tau mRNAs in sections of rat cerebellum. The hybridization signal was consistent with the known localization of brain tau mRNAs, showing the validity of cDNA probes labelled by digoxigenin during the PCR. Using these probes, anin situ hybridization protocol was established and optimized for the localization of tau-related mRNAs in sections of pancreas. The aim was to determine whether these mRNAs were expressed in the exocrine or the endocrine part of the pancreas. A positive signal was found only in the exocrine part of the pancreas, and was distributed exclusively in the cytoplasm of acinar cells. The results described here are the first evidence for a specific expression of tau-related proteins in the exocrine pancreas.     

Plasma membrane transport systems in higher plants: From black boxes to molecular physiology

Considerable progress in identifying transport systems of the plant plasma membrane has been made recently. The putative systems cloned to date comprise H⁺-ATPases, potassium, chloride and water channels, and carriers involved in the transport of glucose, sucrose, amino acids, peptides, potassium, nitrate, ammonium, phosphate, sulfate, iron and copper. Most of these systems were identified first in Arabidopsis thaliana . Successful cloning strategies have involved the following variety of techniques: complementation of yeast mutants, screening of Arabidopsis mutants, immunoscreening of a cDNA expression library expressed in mammalian cells, screening of genomic and cDNA libraries with probes (or degenerate oligonucleotides) derived from yeast and/or animal genes, or database screening for sequence similarity to eukaryotic counterparts. Many related transport systems have subsequently been identified either by screening libraries directly, or by systematic cDNA sequencing programs. Surprisingly large gene families have been revealed. Heterologous expression systems, such as yeast, Xenopus oocytes or insect cells, provide tools for studying the transport activities, biochemical properties and structure-function relationships of these systems. Their expression and functions in planta are investigated using northern blot analysis, in situ hybridization, and transgenic approaches. Individual systems encoded by the same gene family can differ in their transport properties and have distinct tissue expression patterns. Such diversity might be central to the integration of solute transport at the whole plant level, allowing the differential expression of sets of transport systems specifically tailored to the requirements of each tissue.     

Three cDNA clones encoding mouse transplantation antigens: Homology to immunoglobulin genes

We constructed cDNA libraries from poly(A)⁺ RNA isolated from cell lines of two different inbred strains of mice, and screened the libraries with a cDNA clone encoding a human transplantation antigen. Three cDNA clones were identified, sequenced and found to encode amino acid sequences highly homologous to portions of a known mouse transplantation antigen. Comparison of the cDNA sequences of mouse transplantation antigens with the constant region domains of the mouse immunoglobulin μ gene reveals a striking homology, which suggests that the two genes share a common ancestor. Antibody genes undergo DNA rearrangements during B cell differentiation that are correlated with their expression. In contrast, DNA blots with these cDNA probes suggest that the genes for the transplantation antigens are not rearranged in the genomes of liver or embryo cells, which express these antigens, as compared with sperm cells, which do not express these antigens. In Bam HI-digested liver DNAs from different inbred strains of mice, 10–15 bands of hybridization were found. Accordingly, the genes encoding the transplantation antigens appear to constitute a multigene family with similar gene numbers in different mice.