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K U Sprague

Publications and source records attributed to K U Sprague.

32 records · Page 2Linked to original sources
PubMed1984

In vitro transcription of a silkworm 5S RNA gene requires an upstream signal.

The RNA polymerase III transcription complex from Bombyx mori silkworms, in contrast to that from Xenopus frogs, requires a signal that is upstream from the transcription initiation site. We have found that part of the normal 5' flanking sequence is essential for transcription of Bombyx 5S RNA genes by homologous cell-free extracts. The critical region is structurally similar to the upstream signal that directs Bombyx tRNA gene transcription. The association of such a control element with two classes of polymerase III templates argues that it acts through a general component of the Bombyx transcription apparatus.

Animals↗
PubMed1983

A short 5' flanking region containing conserved sequences is required for silkworm alanine tRNA gene activity.

Using partially deleted genes, we have identified an upstream control signal required for transcription of a Bombyx mori (silkworm) tRNA(2) (Ala) gene. The 5' boundary of this essential region lies between 34 and 11 nucleotides preceding the transcription initiation site. Sequences in the 5' half of the tRNA(2) (Ala) coding region are also important. Both regions contain oligonucleotides that are conserved among several Bombyx genes transcribed by RNA polymerase III, suggesting that these sequences may have a general control function.

Journal Article↗
PubMed1982

Silkworm 5S RNA and alanine tRNA genes share highly conserved 5' flanking and coding sequences.

A fragment of Bombyx mori genomic DNA containing one tRNA2Ala gene and one 5S RNA gene has been used to compare the structural features of silkworm 5S RNA and tRNA genes. The nucleotide sequences of both genes and of the primary transcripts produced from them in homologous in vitro transcription systems have been determined. Comparison of the sequences of these two genes with that of another previously analyzed B. mori tRNA2Ala gene reveals common oligonucleotides which may be important transcriptional signals. The oligonucleotides TA(C)TAT, AATTTT, and TTC are located approximately (+/- 1 nucleotide) 29, 19, and 3 nucleotides, respectively, before the transcription initiation sites of the two tRNA2Ala genes and the one 5S RNA gene we have analyzed. The sequence GGGCGTAG(C)TCAG lies within the coding regions of all three genes. The functional significance of these sequences is suggested by their location within regions required for the transcription of silkworm alanine tRNA genes in vitro.

5' Flanking Region↗
PubMed1980

5' flanking sequence signals are required for activity of silkworm alanine tRNA genes in homologous in vitro transcription systems.

We have used homologous in vitro transcription to examine the nucleotide sequences required for activity of cloned Bombyx mori tRNA2Ala genes. We have compared the transcriptional properties of an intact gene and a truncated derivative of the same gene lacking all but 11 nucleotides of normal 5' flanking DNA, and we find that at least two regions of DNA are required for accurate transcription of tRNA2Ala genes by homologous B. mori extracts. One of these sites is retained in the truncated gene and may be within the tRNA coding sequence. Competition experiments with intact and truncated genes indicate that this site probably acts by binding a factor necessary for specificity in polymerase III-catalyzed transcription. The second required site is removed by the Hind III cleavage used to produce shortened genes and thus must be 11 nucleotides or more upstream from the transcription initiation point. Possible roles for this site are discussed.

Alanine↗
PubMed1979

The nucleotide sequence adjacent to poly(A) in silk fibroin messenger RNA.

The 3'-terminal nucleotide sequence of fibroin mRNA has been determined. A cDNA transcript primed by oligo(dT) hybridized to the poly(A) segment of fibroin mRNA was used to determine the sequence immediately adjacent to poly(A). A specific primer complementary to this sequence then allowed synthesis of 5'-terminally homogeneous transcripts amenable to analysis by rapid sequencing methods. The 3' terminus of fibroin mRNA contains an unusually long oligo(U) stretch, as well as the sequence A2UA3 recently found in 3' non-coding regions of other eukaryotic mRNAs. It lacks the oligonucleotides characteristic of the repetitive coding region of fibroin mRNA.

Animals↗
PubMed1979

Alleles of the fibroin gene coding for proteins of different lengths.

Bombyx mori silkworms producing fibroin proteins of different lengths have been analyzed genetically and shown to possess variant alleles of a single fibroin gene. The structures of two alleles have been compared by using restriction endonuclease sites inside and outsite the fibroin gene as physical markers. We find that fibroins distinguishable on the basis of length are encoded by genes with different internal structures and overall lengths. Our results strongly support the idea that rearrangements within the highly repetitive sequences of the fibroin gene are the result of unequal recombination, and can give rise to variant fibroin genes with altered coding lengths.

Alleles↗
PubMed1979

The primary transcription product of a silkworm alanine tRNA gene: identification of in vitro sites of initiation, termination and processing.

A 13.5 Kb fragment of Bombyx mori DNA containing a single tRNA2Ala gene has been cloned, and transcribed in vitro with Xenopus germinal vesicle extracts. The primary transcription product of the tRNA2Ala gene has been isolated and shown to possess an unprocessed triphosphorylated 5' terminus. Products resulting from processing of this transcript have also been isolated and characterized. Complete nucleotide sequence analysis of this cloned alanine tRNA gene and its primary transcript shows that transcription initiates three nucleotides away from the mature tRNA2Ala 5' end and terminates in a U cluster 22 nucleotides beyond the last encoded 3' nucleotide of the mature species. Sequence determination of the products of in vitro maturation shows that in contrast to the tRNA processing mechanism characteristic of procaryotes, the extra 3'-nucleotides in this silkworm tRNA precursor are removed by a single endonucleolytic cleavage.

Alanine↗
PubMed1978

A single base-pair change creates a Chi recombinational hotspot in bacteriophage lambda.

X4+ mutations, responsible for the Chi phenotype in phage lambda, locally increase the rate of recombination promoted by the Escherichia coli recombination system (Rec). X+ mutations in the cII gene, one of a few sites in lambda at which such mutations arise, were located genetically and physically with overlapping deletions. DNA sequence analysis of the deletion segment containing the X+ C mutations showed that two independent X+ C mutations arose by the same A-T to T-A transversion. Presumably, this change creates a nucleotide sequence recognized by a protein involved in a rate-limiting step of recombination.

Base Sequence↗
PubMed1977

The nucleotide sequence of two silk gland alanine tRNAs: implications for fibroin synthesis and for initiator tRNA structure.

The nucleotide sequences of two major alanine tRNAs from the Bombyx mori posterior silk gland have been determined. One of these tRNAs appears to be specific to the silk gland, where its accumulation is associated with the rapid production of fibroin. Both sequences are identical, with the exception of a single nucleotide in the anticodon stem. A striking feature of both alanine tRNAs is that loop IV contains sequences previously believed to be restricted to initiator tRNA.

Alanine↗
PubMed1975

The Bombyx mori silk proteins: characterization of large polypeptides.

Proteins taken directly from the Bombyx mori silk gland have been separated and identified as either fibroin or sericin on the basis of their location within the gland and their amino acid composition. Molecular weights of these polypeptides have been determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and by agarose-guanidine chromatography. Fibroin consists of approximately equimolar amounts of two large (350,000) polypeptide chains. These may be the products of distinct fibroin alleles present in hybrid silkworm strains. Sericin, on the other hand, is composed of at least the three largest polypeptides (130,000-220,000) present in a mixture of proteins ranging in size from about 20,000 to 220,000.

Alleles↗
PubMed1975

The terminal sequences of Bombyx mori 18S ribosomal RNA.

The 5' and 3' terminal T1 oligonucleotides of 32p-labelled B. mori 18S ribosomal RNA were isolated by a two dimensional electrophoretic (diagonal) technique. Nucleotide sequence analysis showed that the 3' terminal fragment, (G)AUCAUUAOH, is identical to that previously obtained from the 18S rRNA of several other eukaryotic species. The sequence of the B. mori 5' terminal fragment is pUCCUCG.

Animals↗
PubMed1975

The 3' terminal oligonucleotide of E. coli 16S ribosomal RNA: the sequence in both wild-type and RNase iii- cells is complementary to the polypurine tracts common to mRNA initiator regions.

Application of Sanger techniques to the analysis of the 3' terminal oligonucleotide from E. coli 32-P-labelled 16 S rRNA yields the sequence AUCACCUCCUUAOH. This sequence is identical in RNA isolated from two wild-type strains (MRE600 and E. coli B, SY106) and from a mutant strain (AB301/105) defective in RNase III. Data presented here explains the previous derivation of an incorrect sequence (AUCCUCACUUCAOH) by others. The functional significance of complementarity between the 3' terminus of 16S rRNA and poly-purine tracts commonly found in mRNA initiator regions is discussed.

Base Sequence↗