Differential gene expression in CD3e- and RAG1-deficient thymuses: definition of a set of genes potentially involved in thymocyte maturation

 A set of 3000 mouse thymus cDNAs was analyzed by extensive measurement of expression using complex-probe hybridization of DNA arrays ("quantitative differential screening"). The complex probes were initially prepared using total thymus RNA isolated from C57BL/6 wild-type (WT), CD3e- and RAG1-deficient mice. Over 100 clones displaying over- or under-expression by at least a factor of two between WT and knockout (KO) thymuses were further analyzed by measuring hybridization signatures with probes from a wide range of KO thymuses, cell types, organs, and embryonic thymuses. A restricted set of clones was selected by virtue of their expression spectra (modulation in KO thymuses and thymocytes, lymphoid cell specificity, and differential expression during embryonic thymus development), sequenced at one extremity, and compared to sequences in databases. Clones corresponding to previously identified genes (e.g., Tcrβ, Tcf1 or CD25) showed expression patterns that were consistent with existing data. Ten distinct clones corresponding to new genes were subjected to further study: Northern blot hybridization, in situ hybridization on thymus sections, and partial or complete mRNA sequence determination. Among these genes, we report a new serine peptidase highly expressed in cortical epithelial cells that we have named thymus-specific serine peptidase (TSSP), and an acidic protein expressed in thymocytes and of unknown function that we have named thymus-expressed acidic protein (TEAP). This approach identifies new molecules likely to be involved in thymocyte differentiation and function. Received: 3 June 1999 / Revised: 3 August 1999     

Characterization and application of soybean YACs to molecular cytogenetics

Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes. In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH). We have screened such a library for low-copy-number clones by hybridization to total genomic DNA. Four clones were chosen for chromosome tagging based upon their low or moderate signal. By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei. FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci. The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes. We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.     

Heterogeneity of polyadenylation site of mRNA coding for human serum albumin

Human fetal liver cDNA was cloned in pBR322 vector by dG:dC-tailing method. The cDNA library was screened for liver-specific clones by means of differential hybridization. Human fetal liver and human kidney cDNAs were used as hybridization probes. Application of this procedure revealed twenty five liver-specific clones among about one thousand recombinants analysed. These clones represent cDNAs corresponding abundant mRNAs. Eighteen clones were identified as encoding serum albumin. Two different mRNA polyadenylation sites were found in four sequenced plasmids. Cleavage/polyadenylation site in two plasmids, pHA1 and pHA12, is situated fifteen nucleotides downstream the AATAAA signal; in two other plasmids, pHA8 and pHA25, this site is ten nucleotides downstream the same signal.     

Isolation of carp cDNA clones,representing developmentally-regulated genes,using a subtractive-hybridization strategy

A subtractive-hybridization technique, combined with differential screenings and subsequent whole mount in situ hybridization (ISH) reactions, was used to isolate novel cDNA clones representing developmentally-regulated genes of carp. Small-scale differential screenings of an oocyte and a segmentation-stage cDNA library using oocyte-specific and segmentation stage-specific enriched probes, yielded 75 positive clones. ISH screening showed that 65% (15) of the oocyte-stage clones and 50% (26) of the segmentation-stage clones were indeed stage-specific. Partial sequence analysis suggests that approximately 65% of the 41 stage-specific clones represent novel genes. In addition, an Otxl clone was isolated. Two novel clones and the Otxl clone are of special interest for developmental studies. The clones represent genes that are locally expressed during embryonic development. The expression patterns of Otxl and one of the novel clones suggest functions in specification of the anterior-posterior axis. The three clones provide molecular markers for the study of gastrulation and the patterning of the a-p axis in teleosts.     

Molecular cloning of genes related to aflatoxin biosynthesis by differential screening.

A differential hybridization strategy was used to clone genes associated with aflatoxin biosynthesis. A genomic library, formed between nuclear DNA and the pUC19 plasmid, was screened with three different cDNA probes by the colony hybridization procedure. Nineteen clones were selected; all were positively correlated with and presumably enriched with genes associated with aflatoxin production. Some of these clones were further characterized by using them as probes in Northern (RNA blot) hybridizations. Five clones hybridized strongly with some polyadenylated RNAs formed during the transition to or during idiophase when aflatoxin was produced. However, little or no corresponding hybridization occurred with polyadenylated RNAs formed in early and mid-log growth phase. Two of the clones were further used as probes to hybridize with polyadenylated RNAs formed under aflatoxin-permissive and nonpermissive temperatures. Hybridization occurred with RNA species formed under the permissive temperature only.     

Molecular cloning of genes related to aflatoxin biosynthesis by differential screening.

A differential hybridization strategy was used to clone genes associated with aflatoxin biosynthesis. A genomic library, formed between nuclear DNA and the pUC19 plasmid, was screened with three different cDNA probes by the colony hybridization procedure. Nineteen clones were selected; all were positively correlated with and presumably enriched with genes associated with aflatoxin production. Some of these clones were further characterized by using them as probes in Northern (RNA blot) hybridizations. Five clones hybridized strongly with some polyadenylated RNAs formed during the transition to or during idiophase when aflatoxin was produced. However, little or no corresponding hybridization occurred with polyadenylated RNAs formed in early and mid-log growth phase. Two of the clones were further used as probes to hybridize with polyadenylated RNAs formed under aflatoxin-permissive and nonpermissive temperatures. Hybridization occurred with RNA species formed under the permissive temperature only.     

In situ hybridization analysis of the temporospatial expression of the midkine/pleiotrophin family in rat embryonic pituitary gland

Pituitary gland development is controlled by numerous signaling molecules, which are produced in the oral ectoderm and diencephalon. A newly described family of heparin-binding growth factors, namely midkine (MK)/pleiotrophin (PTN), is involved in regulating the growth and differentiation of many tissues and organs. Using in situ hybridization with digoxigenin-labeled cRNA probes, we detected cells expressing MK and PTN in the developing rat pituitary gland. At embryonic day 12.5 (E12.5), MK expression was localized in Rathke’s pouch (derived from the oral ectoderm) and in the neurohypophyseal bud (derived from the diencephalon). From E12.5 to E19.5, MK mRNA was expressed in the developing neurohypophysis, and expression gradually decreased in the developing adenohypophysis. To characterize MK-expressing cells, we performed double-staining of MK mRNA and anterior pituitary hormones. At E19.5, no MK-expressing cells were stained with any hormone. In contrast, PTN was expressed only in the neurohypophysis primordium during all embryonic stages. In situ hybridization clearly showed that MK was expressed in primitive (immature/undifferentiated) adenohypophyseal cells and neurohypophyseal cells, whereas PTN was expressed only in neurohypophyseal cells. Thus, MK and PTN might play roles as signaling molecules during pituitary development.     

Analysis of fibrinogen genes in patients with congenital afibrinogenemia

Several cDNA clones coding for A alpha, B beta and gamma chains of fibrinogen have been isolated from a human liver cDNA library. They were selected by differential hybridization with probes raised against fractionated liver mRNA (positive probes) and muscle and albumin mRNA (negative probes), then firmly identified by positive hybridization selection. Three of these clones, encoding A alpha, B beta and gamma fibrinogen chain sequences, were further characterized by restriction mapping and used as probes to characterize fibrinogen mRNAs from adult and fetal liver and fibrinogen genes in normal individuals and two afibrinogenemic patients. The results indicate that there is a single copy of the fibrinogen genes which are present and grossly intact in afibrinogenemic DNA.     

Chromosomal in situ hybridization with double-labeled DNA: signal amplification at the probe level.

A double-labeling approach was applied to nonisotopic in situ hybridization with individual cosmid and plasmid clones, using digoxigenin or biotin as label and a combination of two separate enzymatic labeling methods. Probe labeling was achieved by nick translation, followed by tailing of the probe by terminal deoxynucleotidyl transferase. The double-labeling method, in conjunction with an improved detection protocol, provides for a higher signal intensity than that obtainable with single-labeled probes.     

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 expression and genomic organization of randomly selected cloned Drosophila melanogaster genes

A lambda recombinant DNA library containing Drosophila melanogaster nuclear DNA inserts was screened with cDNA made from oocyte and gastrula poly(A)+ RNA. 124 clones were isolated which represented sequences complementary to a distribution of abundancies of their RNAs. The clone set was then used as probes to identify those whose RNA abundancies changed during embryonic development. The vast majority of clones showed little difference during development. Four different clones were identified whose poly(A)+ RNAs were quantitatively regulated; two were oocyte-specific, and two were embryonic-specific. 44 clones were chosen for in situ hybridization to salivary gland polytene chromosomes. The location and distribution of their sites are described. A class of clones, identified by in situ hybridization to the nucleolus, is further described. These clones contain a scrambled array of ribosomal intervening sequences.     

The space-time distribution of the mRNA of the nuclear proteins c-myc and P-53 in the development of the clawed toad studied by hybridization in situ]

We studied distribution of mRNA for nuclear protooncogene c-myc and nuclear protein P-53 in mature oocytes and embryos of Xenopus laevis from the stage of fertilization up to the stage of hatching by in situ hybridization with histological sections. mRNA for c-myc was present in all cells of the embryo at all studied developmental stages. Between the stage of fertilization and up to the late blastula, mRNA concentration for c-myc decreased progressively in all embryonic cells. During gastrulation a local increase in the concentration of this messenger was found in dorsal mesoderm and ectoderm. At the stage of neurula increased concentration of mRNA for c-myc was observed in all cells of the embryo but the hybridization signal increased particularly distinctly in cells of the neural tube. In studies of P-53 mRNA distribution hybridization signal was detected only in brain cells after stage 20 of development (after closure of the neural folds) and up to the stage of hatching.