A Polymerase-chain-reaction Assay for the Specific Identification of Transcripts Encoded by Individual Carcinoembryonic Antigen (CEA)-gene-family Members

Carcinoembryonic antigen (CEA) is a tumor marker that belongs to a family of closely related molecules with variable expression patterns. We have developed sets of oligonucleotide primers for the specific amplification of transcripts from individual CEA-family members using the reverse transcriptase/ polymerase chain reaction (RT/PCR). Specific primer sets were designed for CEA, non-specific cross-reacting antigen (NCA), biliary glycoprotein (BGP), carcinoembryonic antigen gene-family members 1, 6 and 7 (CGMI, CGM6 and CGM7), and one set for all pregnancy-specific glycoprotein (PSG) transcripts. Primers were first tested for their specificity against individual cDNA clones and product-hybridization with internal, transcript-specific oligonucleotides. Total RNA from 12 brain and 63 gynecological tumors were then tested for expression of CEA-related transcripts. None were found in tumors located in the brain, including various mesenchymal and neuro-epithelial tumors. CEA and NCA transcripts were, however, present in an adenocarcinoma located in the nasal sinuses. In ovarian mucinous adenocarcinomas, we always found co-expression of CEA and NCA transcripts, and occasionally BGP mRNA. CEA-related transcripts were also found in some serous, endometrioid and clear-cell ovarian carcinomas. CEA, NCA and BGP transcripts were present in endometrial carcinomas of the uterus and cervical carcinomas, whereas uterine leiomyomas were completely negative. No transcripts were found from CGM 1, CGM6, CGM7 or from PSG genes in any of the tumors tested. The PCR data were compared with immunohistochemical investigations of ovarian tumors at the protein level using CEA (26/3/13)-, NCA-50/90 (9A6FR) and NCA-95 (80H3)-specific monoclonal antibodies

Broad application of a simple and affordable protocol for isolating plant RNA

BACKGROUND: Standard molecular biological methods involve the analysis of gene expression in living organisms under diverse environmental and developmental conditions. One of the most direct approaches to quantify gene expression is the isolation of RNA. Most techniques used to quantify gene expression require the isolation of RNA, usually from a large number of samples. While most published protocols, including those for commercial reagents, are either labour intensive, use hazardous chemicals and/or are costly, a previously published protocol for RNA isolation in Arabidopsis thaliana yields high amounts of good quality RNA in a simple, safe and inexpensive manner. FINDINGS: We have tested this protocol in tomato and wheat leaves, as well as in Arabidopsis leaves, and compared the resulting RNA to that obtained using a commercial phenol-based reagent. Our results demonstrate that this protocol is applicable to other plant species, including monocots, and offers yield and purity at least comparable to those provided by commercial phenol-based reagents. CONCLUSIONS: Here, we show that this previously published RNA isolation protocol can be easily extended to other plant species without further modification. Due to its simplicity and the use of inexpensive reagents, this protocol is accessible and affordable and can be easily implemented to work on different plant species in laboratories worldwide

Neoplastic transformation of mouse C3H 10T1/2 and Syrian hamster embryo cells by heavy ions

C3H 10T1/2 mouse-embryo fibroblasts were used for transformation experiments to study the effectiveness of various heavy ions with energies up to 20 MeV/u and LET values from 170 to 16.000 keV/μm. The transformation frequency per unit absorbed dose decreased with increasing ionization density; at the highest values of LET we found a decrease even of the transformation efficiency per unit fluence. Uranium ions at energies of 5, 9, and 16.3 MeV/u did not induced any transformation. In additional studies piimary Syrian hamster embryo cells (SHE) were exposed to heavy ions in order to characterize cytological and molecular changes which may be correlated with neoplastic transformation. Growth behaviour, chromosomal status, tumorigenicity in nude mice, and expression of oncogenes of transformed cell lines were examined

The Human Pregnancy-Specific Glycoprotein Genes are Tightly Linked on the Long Arm of Chromosome 19 and are Coordinately Expressed

The pregnancy-specific glycoprotein (PSG) genes encode a group of proteins which are found in large amounts in placenta and maternal serum. In situ hybridization analyses of metaphase chromosomes reveal that all the human pregnancy-specific glycoprotein (PSG) genes are located on the long arm of chromosome 19 (19q13.2–13.3), overlapping the region containing the closely-related carcinoembryonic antigen (CEA) gene subgroup. Higher resolution analyses indicate that the PSG genes are closely linked within an 800kb SacII restriction endonuclease fragment. This has been confirmed through restriction endonuclease mapping and DNA sequence analyses of isolated genomic clones, which show that at least some of these genes are located in very close proximity. Further, these studies have helped to identify a new member of the PSG gene sub-family (PSG7). DNA/RNA hybridization analyses, using gene-specific oligonucleotide probes based on published sequences, showed that five from six PSG genes tested are coordinately transcribed in the placenta. Due to the close proximity of these genes and their coordinated expression pattern, common transcriptional regulatory elements may exist

HLA-G: expression in human keratinocytes in vitro and in human skin in vivo

Classical, polymorphic major histocompatibility complex class I molecules are expressed on most nucleated cells.They present peptides at the cell surface and, thus, enable the immune system to scan peptides for their antigenicity. The function of the other, nonclassical class I molecules in man is controversial. HLA-G which has been shown by transfection experiments to be expressed at the cell surface, is only transcribed in placental tissue and in the fetal eye.Therefore, a role of HLA-G in the control of rejection of the allogeneic fetus has been discussed. We found that HLA-G expression is induced in keratinocytes by culture in vitro. Three different alternative splicing products of HLA-G can be detected: a full length transcript, an mRNA lacking exon 3 and a transcript devoid of exon 3 and 4. Reverse transcription followed by polymerase chain reaction also revealed the presence of HLA-G mRNA in vivo in biopsies of either diseased or healthy skin

Identification of an osteocalcin isoform in fish with a large acidic prodomain

Osteocalcin is a small, secreted bone protein whose gene consists of four exons. In the course of analyzing the structure of fish osteocalcin genes, we recently found that the spotted green pufferfish has two possible exon 2 structures, one of 15 bp and the other of 324 bp. Subsequent analysis of the pufferfish cDNA showed that only the transcript with a large exon 2 exists. Exon 2 codes for the osteocalcin propeptide, and exon 2 of pufferfish osteocalcin is ∼3.4-fold larger than exon 2 previously found in other vertebrate species. We have termed this new pufferfish osteocalcin isoform OC2. Additional studies showed that the OC2 isoform is restricted to a unique fish taxonomic group, the Osteichthyes; OC2 is the only osteocalcin isoform found so far in six Osteichthyes species, whereas both OC1 and OC2 isoforms coexist in zebrafish and rainbow trout. The larger size of the OC2 propeptide is due to an acidic region that is likely to be highly phosphorylated and has no counterpart in the OC1 propeptide. We propose 1) that OC1 and OC2 are encoded by distinct genes that originated from a duplication event that probably occurred in the teleost fish lineage soon after divergence from tetrapods and 2) that the novel OC2 propeptide could be, if secreted, a phosphoprotein that participates in the regulation of biomineralization through its large acidic and phosphorylated propeptide

Hypertension in mice lacking 11beta-hydroxysteroid dehydrogenase type 2

Deficiency of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) in humans leads to the syndrome of apparent mineralocorticoid excess (SAME), in which cortisol illicitly occupies mineralocorticoid receptors, causing sodium retention, hypokalemia, and hypertension. However, the disorder is usually incompletely corrected by suppression of cortisol, suggesting additional and irreversible changes, perhaps in the kidney. To examine this further, we produced mice with targeted disruption of the 11β-HSD2 gene. Homozygous mutant mice (11β-HSD2(–/–)) appear normal at birth, but ∼50% show motor weakness and die within 48 hours. Both male and female survivors are fertile but exhibit hypokalemia, hypotonic polyuria, and apparent mineralocorticoid activity of corticosterone. Young adult 11β-HSD2(–/–) mice are markedly hypertensive, with a mean arterial blood pressure of 146 ± 2 mmHg, compared with 121 ± 2 mmHg in wild-type controls and 114 ± 4 mmHg in heterozygotes. The epithelium of the distal tubule of the nephron shows striking hypertrophy and hyperplasia. These histological changes do not readily reverse with mineralocorticoid receptor antagonism in adulthood. Thus, 11β-HSD2(–/–) mice demonstrate the major features of SAME, providing a unique rodent model to study the molecular mechanisms of kidney resetting leading to hypertension. J. Clin. Invest. 103:683–689 (1999

Cell arrest and cell death in mammalian preimplantation development

The causes, modes, biological role and prospective significance of cell death in preimplantation development in humans and other mammals are still poorly understood. Early bovine embryos represent a very attractive experimental model for the investigation of this fundamental and important issue. To obtain reference data on the temporal and spatial occurrence of cell death in early bovine embryogenesis, three-dimensionally preserved embryos of different ages and stages of development up to hatched blastocysts were examined in toto by confocal laser scanning microscopy. In parallel, transcript abundance profiles for selected apoptosis-related genes were analyzed by real-time reverse transcriptase-polymerase chain reaction. Our study documents that in vitro as well as in vivo, the first four cleavage cycles are prone to a high failure rate including different types of permanent cell cycle arrest and subsequent non-apoptotic blastomere death. In vitro produced and in vivo derived blastocysts showed a significant incidence of cell death in the inner cell mass (ICM), but only in part with morphological features of apoptosis. Importantly, transcripts for CASP3, CASP9, CASP8 and FAS/FASLG were not detectable or found at very low abundances. In vitro and in vivo, errors and failures of the first and the next three cleavage divisions frequently cause immediate embryo death or lead to aberrant subsequent development, and are the main source of developmental heterogeneity. A substantial occurrence of cell death in the ICM even in fast developing blastocysts strongly suggests a regular developmentally controlled elimination of cells, while the nature and mechanisms of ICM cell death are unclear. Morphological findings as well as transcript levels measured for important apoptosis-related genes are in conflict with the view that classical caspase-mediated apoptosis is the major cause of cell death in early bovine development

A Novel Mutation in the Upstream Open Reading Frame of the CDKN1B Gene Causes a MEN4 Phenotype

PubMed ID: 23555276This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited