A universal primer mixture for sequence determination at the 3' ends of cDNAs

We have devised a universal primer which can be used to sequence the 3'-ends of cloned cDNAs containing a polyA tail. The primer consists of an equimolar mixture of three primers: 20 T nucleotides followed by either an A, C, or G nucleotide (5'----3'). With this primer mixture and the dideoxynucleotide chain termination method, we determined the 3'-terminal sequence of human beta-actin cDNA in an Okayama-Berg vector, in four parallel sets of reactions containing either a single primer (T20G, T20C, or T20A) or an equimolar mixture of all three primers. Priming with both T20A and the triple mixture gave clearly readable results that agree with the known sequence of the human beta-actin gene, and we have applied this method successfully to several other cDNAs in the Okayama-Berg expression vector. Use of this universal primer mixture facilitates determination of sequences at the 3'-ends of cDNAs while by-passing the polyA tail region.     

Functional tRNAs with altered 3' ends.

The CCA trinucleotide is a universally conserved feature of the 3' end of tRNAs, where it serves as the site of amino acid attachment. Despite this extreme conservation, we have isolated functional mutants of tRNA(His) and tRNA(Val1) with altered CCA ends. A mutant that leads to de-repression of the histidine biosynthetic operon in Salmonella typhimurium has been characterized and found to have the CCA end of the sole tRNA(His) species mutated to UCA. However, constructed mutants of tRNA(His) with ACA or GCA ends appeared to be nonfunctional in vivo. Mutants of Escherichia coli tRNA(Val1) with GCA or ACA ends were isolated on the basis of their ability to promote frameshifting at a specific sequence. These same tRNA(Val1) mutants also caused read-through of stop codons that were one, or in some instances two, codons downstream of the valine codon decoded by the mutant tRNA. A startling implication of these data is that disruption of interactions between the CCA end of the tRNA and the large ribosomal subunit promotes these aberrant codon-anticodon interactions on the small ribosomal subunit.     

Interaction of the mouse chromosomal protein HMG-I with the 3' ends of genes in vitro

High affinity mouse HMG-I binding sites have been distinguished from other (A+T)-rich sequences using band competition assays. These sites have been found 3' to the coding regions of a variety of genes. For the herpes simplex virus thymidine kinase and minute virus of mice genes, high affinity HMG-1 binding sites were further localized to the functional polyadenylation signal by DNase I footprinting. These results suggest that HMG-I may function at the 3' ends of genes in vivo.     

Integrons found in different locations have identical 5' ends but variable 3' ends.

The positions of the outer boundaries of the 5'- and 3'-conserved segment sequences of integrons found at several different locations have been determined. The position of the 5' end of the 5'-conserved segment is the same for six independently located integrons, In1 (R46), In2 (Tn21), In3 (R388), In4 (Tn1696), In5 (pSCH884), and In0 (pVS1). However, the extent of the 3'-conserved segment differs in each integron. The sequences of In2 and In0 diverge first from the conserved sequence, and their divergence point corresponds to the 3'-conserved segment endpoint defined previously (H.W. Stokes and R.M. Hall, Mol. Microbiol. 3:1669-1683, 1989), which now represents the endpoint of a 359-base deletion in In0 and In2. The sequence identity in In3, In1, In4, and In5 extends beyond this point, but each sequence diverges from the conserved sequence at a different point within a short region. Insertions of IS6100 were identified adjacent to the end of the conserved region in In1 and 123 bases beyond the divergence point of In4. These 123 bases are identical to the sequence found at the mer end of the 11.2-kb insertion in Tn21 but are inverted. In5 and In0 are bounded by the same 25-base inverted repeat that bounds the 11.2-kb insert in Tn21, and this insert now corresponds to In2. However, while In0, In2, and In5 have features characteristic of transposable elements, differences in the structures of these three integrons and the absence of evidence of mobility currently preclude the identification of all of the sequences associated with a functional transposon of this type.     

A new method for the mapping of 5' ends of RNAs

In this article, we describe a new procedure to map 5′ ends of RNAs. The procedure consists in the use of specific RNase H digestion of a hybrid formed by the RNA and a complementary DNA oligonucleotide. Northern blot hybridization of the resulting RNA fragment allows an accurate measurement of its length. Although we generally use this procedure as a control of previously performed primer extension analyses, the absence of nonspecific bands, which often occur in primer extensions on RNA templates with extended secondary structures, suggests that our method may be preferable when these difficult templates are analyzed.     

Shaw-like rat brain potassium channel cDNA's with divergent 3' ends

The complete amino acid sequence of a potassium channel protein of rat brain, Kv3.2b, plus a partial sequence of a related channel, Kv3.2c, are deduced from molecular cloning of the respective cDNA's. Kv3.2b and Kv3.2c share extensive amino acid sequence identity with a previously identified channel, RKShIIIA[1], before diverging to unique carboxy termini. Probes specific for Kv3.2b and RKShIIIA detect similarly sized mRNA's on Northern blots. These two proteins are encoded by a single gene based on genomic Southern blotting, and therefore arise by alternative splicing. In vitro transcribed mRNA for Kv3.2b induces the expression of outward K+ currents in Xenopus oocytes under voltage-clamp conditions.     

Telomerase is required to protect chromosomes with vertebrate-type T2AG3 3' ends in Saccharomyces cerevisiae

Telomeres containing vertebrate-type DNA repeats can be stably maintained in Saccharomyces cerevisiae cells. We show here that telomerase is required for growth of yeast cells containing these vertebrate-type telomeres. When present at the chromosome termini, these heterologous repeats elicit a DNA damage response and a certain deprotection of telomeres. The data also show that these phenotypes are due only to the terminal localization of the vertebrate repeats because if they are sandwiched between native yeast repeats, no phenotype is observed. Indeed and quite surprisingly, in this latter situation, telomeres are of virtually normal lengths, despite the presence of up to 50% of heterologous repeats. Furthermore, the presence of the distal vertebrate-type repeats can cause increased problems of the replication fork. These results show that in budding yeast the integrity of the 3' overhang is required for proper termination of telomere replication as well as protection.     

Stability of 3' double nucleotide overhangs that model the 3' ends of siRNA

Thermodynamic parameters are reported for duplex formation in 1 M NaCl for 16 RNA sequences, each containing a core tetramer duplex, GGCC, and a 3' overhang consisting of two bases. The results indicate additional double-helical stability is conferred by the double 3' terminal overhang relative to the single 3' terminal overhang. A nearest-neighbor analysis of the data indicates that the free energy contribution at 37 degrees C of the second base in the double 3' terminal overhang varies from 0 to 0.7 kcal/mol. The second base in the 3' double overhang can contribute nearly the same stability to a duplex as a base pair or a 3' dangling overhang. Stability contribution of a dangling base, two nucleotides removed from the 3' end of a duplex, is dependent upon both the identity of the base as well as that of the dangling base that it neighbors. A second dangling base only increases the stability of the duplex when it is neighboring a 3' purine dangling nucleotide. Furthermore, a second dangling pyrimidine provides a greater contribution to duplex stability than a purine. A nearest-neighbor model was developed to predict the influence of 3' double overhang on the stability of duplex formation. The model improves the prediction of free energy and melting temperature when tested against six sequences with different core duplexes.     

A novel method to produce Anabaena-free Azolla by in vitro fertilization of micromanipulated megasporocarps

In the Azolla-Anabaena azollae symbiotic system, Anabaena akinetes get entrapped between the indusium and the apical cap of the megaspore apparatus during megasporocarp development, thus maintaining the continuity of the cyanobacterial association throughout the life cycle of the fern. The entrapped akinetes serve as the source of inoculum for infecting the new sporophyte when it is emerging from the megaspore apparatus. A procedure to generate Anabaena-free Azolla was developed by fertilizing the germinating megasporocarps in which the indusium along with the akinetes were removed by micromanipulation. This method has the advantage of not requiring drastic treatments of Azolla with antibiotics to eliminate the endosymbiotic cyanobacterial cells. Details of this new method and its usefulness in studies aimed at recombination of Azolla with Anabaena azollae are discussed.Abbreviations IRRI International Rice Research Institute - I^– IRRI medium devoid of combined nitrogen - I⁺ IRRI medium containing combined nitrogen - SDS sodium dodecyl sulfate