Cloning of PCR-amplified total cDNA: construction of a mouse oocyte cDNA library.

We describe a general method for the synthesis and cloning of cDNA, applicable to cases in which the availability of biological material for mRNA extraction is extremely limited. A protocol allowing amplification of a heterogeneous mixture of cDNAs by the polymerase chain reaction has been devised and applied successfully to the construction of an apparently representative cDNA library, using as a model of a scarce RNA source 50 mouse ovulated eggs that can yield a maximum of 1.75 ng of poly(A)+ RNA. However, about 5% of the material obtained after amplification was adequate for cloning. Using the cloned sequences, we have derived a preliminary indirect measurement of the sequence complexity of the maternal poly(A)+ RNA in this mammalian oocyte.     

Assaying the polyadenylation state of mRNAs

The poly(A) tail present at the 3' end of most eukaryotic mRNAs can play a critical role in message translation and stability. Therefore, identifying alterations in poly(A) tail length can yield important insights into an mRNA's function and subsequent physiological impact. Here, we present three methods for assaying polyadenylation of a specific mRNA in the context of total cellular RNA. The first method described, oligo(dT)/RNase H-Northern analysis, is the classic labor-intensive assay for polyadenylation and is included for historical reference and as a potential experimental control for the poly(A) test (PAT) assays described subsequently. The PAT methods-rapid amplification of cDNA ends-PAT (RACE-PAT), and ligase-mediated PAT (LM-PAT)-are polymerase chain reaction-driven assays that allow speed, sensitivity, and length quantitation. The PAT assays can be conducted in a single day and can readily detect the poly(A) status of an mRNA present in subnanogram quantities of total cellular RNA.     

Chum-RNA allows preparation of a high-quality cDNA library from a single-cell quantity of mRNA without PCR amplification

Linear RNA amplification using T7 RNA polymerase is useful in genome-wide analysis of gene expression using DNA microarrays, but exponential amplification using polymerase chain reaction (PCR) is still required for cDNA library preparation from single-cell quantities of RNA. We have designed a small RNA molecule called chum-RNA that has enabled us to prepare a single-cell cDNA library after four rounds of T7-based linear amplification, without using PCR amplification. Chum-RNA drove cDNA synthesis from only 0.49 femtograms of mRNA (730 mRNA molecules) as a substrate, a quantity that corresponds to a minor population of mRNA molecules in a single mammalian cell. Analysis of the independent cDNA clone of this library (6.6 × 10⁵ cfu) suggests that 30-fold RNA amplification occurred in each round of the amplification process. The size distribution and representation of mRNAs in the resulting one-cell cDNA library retained its similarity to that of the million-cell cDNA library. The use of chum-RNA might also facilitate reactions involving other DNA/RNA modifying enzymes whose Michaelis constant (K_m) values are around 1 mM, allowing them to be activated in the presence of only small quantities of substrate.     

Study of transcription of the human cytochrome P450IID6 gene by polymerase chain reaction]

Polymerase chain reaction was used to study the expression of the drug metabolism gene. Primers complementary to the 2070-2090 and 2912-2930 sites within exons 4 and 6 of the gene CYP2D6 were synthesized. The amplification of the cDNA from total human liver mRNA was achieved. The length of the fragment obtained (238 bp) was in accordance with the distance between the primers binding sites in cDNA. The amplification of the DNA from the same source led to the longer fragment due to the presence of introns. The total RNA from the blood cells of the extensive metabolizers was shown to contain the mRNA transcribed from the CYP2D6 gene. The Taq polymerase reaction in the presence of cDNA derived from a poor metabolizer did not lead to the synthesis of the 238 bp fragment.     

Cloning and characterisation of a channel catfish (Ictalurus punctatus) Mx gene

A 2.5 kb full-length cDNA clone of a channel catfish (Ictalurus punctatus) Mx gene was obtained using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) from RNA extracted from the liver of poly I:C stimulated channel catfish. The gene consists of an open reading frame of 1905 nucleotides encoding a 635 amino acid protein. The predicted protein is 72.5 kDa and contains the dynamin family signature, a tripartite GTP binding motif and a leucine zipper, characteristic of all known Mx proteins. The catfish Mx protein exhibited 79% identity with perch Mx and between 71% and 74% identity with the three Atlantic salmon and the three rainbow trout Mx proteins. Mx mRNA was constitutively expressed in channel catfish ovary (CCO) cells, but in higher quantities in response to poly I:C treatment. Mx was induced in channel catfish following injection with channel catfish virus (CCV) and poly I:C.     

Reverse transcription of thyroglobulin 33-S mRNA.

Bovine thyroglobulin 33-S mRNA has been used as a template for the synthesis of a complementary DNA, using RNA-directed DNA polymerase from the avian myeloblastosis virus. The yield of the reaction was relatively poor and the size of the cDNA did not exceed 10 S. Nevertheless, a copy of high specific radioactivity (approximately 10(7) counts. min-1 microgram-1) could be obtained which hybridized specifically back to its template with an rot1/2 value about 5 times higher than that observed in hybridizations between hemoglobin mRNA (alpha + beta chain) and hemoglobin cDNA. This suggests that thyroglobulin mRNA does not contain extensive internal repetitive sequences. Quantification of thyroglobulin mRNA sequences among various RNA preparations from the beef thyroid was performed using cDNA/RNA hybridizations in RNA excess. The results confirmed that thyroglobulin mRNA represents the large majority of mRNA in membrane-bound polysomes and indicated the virtual absence of thyroglobulin sequences on free polyosomes. The cDNA transcribed from mRNA of bovine origin hybridized efficiently with thyroid RNA from goats, dogs and humans. Although the heterologuous hybrids exhibited the expected decrease in thermal stability, the bovine cDNA provides an appropriate probe for studies dealing with the expression of the thyroglobulin gene in various mammals including man.     

Characterization of human poly(ADP-ribose) polymerase with autoantibodies

The addition of poly(ADP-ribose) chains to nuclear proteins has been reported to affect DNA repair and DNA synthesis in mammalian cells. The enzyme that mediates this reaction, poly(ADP-ribose) polymerase, requires DNA for catalytic activity and is activated by DNA with strand breaks. Because the catalytic activity of poly(ADP-ribose) polymerase does not necessarily reflect enzyme quantity, little is known about the total cellular poly(ADP-ribose) polymerase content and the rate of its synthesis and degradation. In the present experiments, specific human autoantibodies to poly(ADP-ribose) polymerase and a sensitive immunoblotting technique were used to determine the cellular content of poly(ADP-ribose) polymerase in human lymphocytes. Resting peripheral blood lymphocytes contained 0.5 X 10(6) enzyme copies per cell. After stimulation of the cells by phytohemagglutinin, the poly(ADP-ribose) polymerase content increased before DNA synthesis. During balanced growth, the T lymphoblastoid cell line CEM contained approximately 2 X 10(6) poly(ADP-ribose) polymerase molecules per cell. This value did not vary by more than 2-fold during the cell growth cycle. Similarly, mRNA encoding poly(ADP-ribose) polymerase was detectable throughout S phase. Poly(ADP-ribose) polymerase turned over at a rate equivalent to the average of total cellular proteins. Neither the cellular content nor the turnover rate of poly(ADP-ribose) polymerase changed after the introduction of DNA strand breaks by gamma irradiation. These results show that in lymphoblasts poly(ADP-ribose) polymerase is an abundant nuclear protein that turns over relatively slowly and suggest that most of the enzyme may exist in a catalytically inactive state.