Evidence that the entire length of a kinetoplast DNA minicircle is transcribed in Trypanosoma cruzi

A 1.3 kb cDNA (cDNA52) was derived from Trypanosoma cruzi trypomastigote mRNA. Using single stranded probes in Northern blots, we identified the putative coding strand of cDNA52. In addition, a minor band was detected in RNA from epimastigotes that was absent in RNA from trypomastigotes. Nucleotide sequence analysis revealed that cDNA52 was highly homologous to T. cruzi kinetoplast DNA minicircle sequences. All four conserved regions of T. cruzi minicircles were identified in cDNA52. Using several criteria, we demonstrated that the hybridization signals were not caused by contaminating minicircle DNA in the RNA preparations. The data provide direct evidence for the unprecedented finding that the entire length of a kDNA minicircle is transcribed in T. cruzi.     

Complex population of nonpolyadenylated messenger RNA in mouse brain

The complexity of nonadenylated mRNA [poly(A)-mRNA] has been determined by hybridization with single-copy DNA (scDNA) and cDNA. Our results show that poly(A)- and poly(A)+ mRNA are essentially nonoverlapping (nonhomologous) sequence populations of similar complexity. The sum of the complexities of poly(A)+ mRNA and poly(A)- mRNA is equal to that of total polysomal RNA or total mRNA, or the equivalent of approximately 1.7 x 10(5) different sequences 1.5 kb in length. Poly(A)- mRNA, isolated from polysomal RNA by benzoylated cellulose chromatography, hybridized with 3.6% of the scDNA, corresponding to a complexity of 7.8 x 10(4) different 1.5 kb sequences. The equivalent of only one adenosine tract of approximately 20 nucleotides per 100 poly(A)- mRNA molecules 1.5 kb in size was observed by hybridization with poly(U). cDNA was transcribed from poly(A)- mRNA using random oligonucleotides as primers. Only 1-2% of the single-copy fraction of this cDNA was hybridized using poly(A)+ mRNA as a driver. These results show that poly(A)- mRNA shares few sequences with poly(A)+ mRNA and thus constitutes a separate, complex class of messenger RNA. These measurements preclude the presence of a complex class of bimorphic mRNAs [that is, species present in both poly(A)+ and poly(A)- forms] in brain polysomes.     

Molecular identification of ADP-ribosylation factor mRNAs and their expression in mammalian cells

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that serve as GTP-dependent allosteric activators of cholera toxin ADP-ribosyltransferase activity. Four species of mammalian ARF, termed ARF 1-4, have been identified by cloning. Hybridization of a bovine ARF 2 cDNA under low stringency with mammalian poly(A)+ RNA resulted in multiple bands that were subsequently assigned to the known ARF genes using ARF-specific oligonucleotide probes. The relative signal intensities of some bands (e.g. the 3.8- and 1.3-kilobase (kb) mRNAs) that hybridized with the cDNA were not, however, consistent with the intensities observed with the individual ARF-specific oligonucleotide probes. These inconsistencies suggested that other ARF-like mRNAs were comigrating with known ARF mRNAs. To explore this possibility, a cyclic AMP-differentiated HL-60 Lambda ZAP library was screened using the bovine ARF 2 cDNA. Clones corresponding to known ARF genes (1, 3, and 4) were identified by hybridization of positive clones with oligonucleotide probes specific for each ARF species; ARF 2 cDNA-positive, oligonucleotide-negative clones were sequenced. Two new ARF-like genes, ARF 5 and 6, encoding proteins of 180 and 175 amino acids, respectively, were identified. Both proteins contain consensus sequences believed to be involved in guanine nucleotide binding and GTP hydrolysis. ARF 5 was most similar in deduced amino acid sequence to ARF 4, which also has 180 amino acids. ARF 6, whose deduced amino acid sequence is identical with that of a putative chicken pseudogene (CPS1) except for a serine/threonine substitution, was different from other ARF species in size and deduced amino acid sequence. With mammalian poly(A)+ RNA from a variety of tissues and cultured cells, ARF 5 preferentially hybridized with a 1.3-kb mRNA, whereas ARF 6 hybridized with 1.8- and 4.2-kb mRNAs. The fact that the sizes of these mRNAs are similar to those of other ARFs (ARF 1, 1.9 kb; ARF 2, 2.6 kb; ARF 3, approximately 3.8 and 1.3 kb; ARF 4, 1.8 kb) explain the previously observed inconsistencies between the cDNA and ARF-specific oligonucleotide hybridization patterns. All six ARF cDNAs are more similar to each other than to other approximately 20-kDa guanine nucleotide-binding proteins.     

Isolation of cDNAs encoding the CD19 antigen of human and mouse B lymphocytes. A new member of the immunoglobulin superfamily

The CD19 (B4) molecule is a m.w. 95,000 cell-surface protein of human B lymphocytes that is expressed before Ig and persists throughout differentiation. In this report, cDNA clones that encode the CD19 molecule were isolated and the amino acid sequence of CD19 was determined. A cDNA clone that selectively hybridized to RNA from CD19+ cell lines was selected from a human tonsilar cDNA library using differential hybridization. This cDNA was used to isolate additional cDNA clones. Four of the five longest cDNA clones isolated were sequenced and found to contain unique sequences presumed to be introns. One clone, pB4-19, was near full length (2.1 kb) and did not contain these putative introns. pB4-19 contained an 1685 bp open reading frame that could encode a protein of about 60 kDa. COS cells that were transfected with pB4-19 expressed a nascent cell surface structure reactive with the anti-B4 antibody. Immunoprecipitation of this structure from surface-iodinated COS cells with the anti-B4 antibody revealed a m.w. 85,000 protein. Northern blot analysis indicated that pB4-19 hybridized with a predominant mRNA species of 2.4 kb and a minor species of 1.5 kb, found in only CD19+ cells. The pre-B cell line, PB-697, also expressed four larger RNA species that hybridized with pB4-19. cDNA clones that encode the putative cytoplasmic portion (247 amino acids) of the mouse CD19 molecule were also isolated and found to be highly homologous (79 and 75%) with the human CD19 nucleotide and amino acid sequences. The deduced amino acid sequence of the CD19 cytoplasmic tail shared no significant homology with other known proteins but the putative extracellular region contained two Ig-like domains indicating that CD19 is a new member of the Ig superfamily.     

Multiple constitutive but phenobarbital-inducible rat hepatic nuclear RNA species homologous to a novel cytochrome P-450 cDNA

A cDNA clone, pPB8, representing partial information for a phenobarbital-inducible rat hepatic cytochrome P-450, immunochemically related to cytochrome P-450b and/or P-450e, hybridized to multiple hepatic nuclear RNA species. In addition to the 3.7 +/- 0.2 kb mRNA encoding this novel cytochrome P-450 isozyme, pPB8 hybridized to nuclear RNAs of 4.9 +/- 0.3, 5.4, 5.7 +/- 0.2, and 6.3 +/- 0.1 kb. These nuclear RNAs were constitutively expressed and were inducible to various extents by phenobarbital administration. The time course of induction of these nuclear RNA components suggested product-precursor relationships. A "full-length" cDNA clone, pPB8/7, synthesized from poly(A)+ RNA homologous to pPB8, detected two mRNA species of 4.6 and 1.8 kb. The 4.6 kb nuclear RNA was inducible by 3-methylcholanthrene, Aroclor 1254, and phenobarbital, while the 1.8 kb nuclear RNA was not appreciably affected. It is suggested that pPB8 and pPB8/7 were synthesized from distinct mRNAs that share homology in their 3' regions.     

cDNA clones encoding bovine interphotoreceptor retinoid binding protein

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.