Nuclear factors in B lymphoma enhance splicing of mouse membrane-bound mu mRNA in Xenopus oocytes.

Regulation of the synthesis of membrane-bound and secreted immunoglobulin mu heavy chains at the level of RNA processing is an important element for B cell development. The precursor mu RNA is either polyadenylated at the upstream poly(A) site (for the secreted form) or spliced (for the membrane-bound form) in a mutually exclusive manner. When the mouse mu gene linked to the SV40/HSV-TK hybrid promoter was microinjected into Xenopus oocytes, the mu messenger RNA (mRNA) was altered by coinjection of nuclei of mouse surface IgM-bearing B-lymphoma cells to include the synthesis of the membrane-bound form. An increase in the membrane-bound form was not observed when nuclei of IgM-secreting hybridoma cells or fibroblast cells were coinjected. Deletion of the upstream poly(A) site did not eliminate the effect of B-lymphoma nuclei suggesting that membrane-specific splicing is stimulated. Further, splicing of other mu gene introns was not affected by coinjection of B-lymphoma nuclei. These results suggest that mature B cells contain one or more transacting nuclear factors that stimulate splicing specific for membrane-bound mu mRNA.

Ras p21 as a potential mediator of insulin action in Xenopus oocytes.

The oncogene protein product (p21) of the ras gene has been implicated in mediating the effects of a variety of growth factors and hormones. Microinjection of monoclonal antibody 6B7, which is directed against a synthetic peptide corresponding to a highly conserved region of p21 (amino acids 29 to 44) required for p21 function, specifically inhibited Xenopus oocyte maturation induced by incubation with insulin. The inhibition was dose-dependent and specific since (i) the same antibody had no effect on progesterone-induced maturation, (ii) immunoprecipitation and Western blotting indicated that the antibody recognized a single protein of molecular weight 21,000 in oocyte extracts, and (iii) inhibition was not observed with identical concentrations of normal immunoglobulin. Thus, p21 appears to be involved in mediating insulin-induced maturation of Xenopus oocytes. Furthermore, the mechanism may involve phosphorylation of p21, as p21 was found to be a substrate of the insulin receptor kinase.

Effects of intron length on differential processing of mouse mu heavy-chain mRNA.

Production of membrane-bound and secreted forms of mouse mu heavy-chain mRNA is controlled by differential processing in a developmental-stage-specific manner. We have analyzed the effects of various deletions and insertions in the C4-M1 intron of the mouse mu gene on the differential processing of mu mRNA. We show that there is a correlation between the length of the C4-M1 intron and the molar ratio of membrane-bound to secreted mu mRNAs, i.e., the shorter the C4-M1 intron, the higher the ratio. Since the poly(A) addition signal in the C4-M1 intron seems to be intact in the mutant mu genes, it is likely that the efficiency of splicing of the C4-M1 intron is affected by changes in the intron length.

Differential order of replication of Xenopus laevis 5S RNA genes.

In Xenopus laevis there are two multigene families of 5S RNA genes: the oocyte-type 5S RNA genes which are expressed only in oocytes and the somatic-type 5S RNA genes which are expressed throughout development. The Xenopus 5S RNA replication-expression model of Gottesfeld and Bloomer (Cell 28:781-791, 1982) and Wormington et al. (Cold Spring Harbor Symp. Quant. Biol. 47:879-884, 1983) predicts that the somatic-type 5S RNA genes replicate earlier in the cell cycle than do the oocyte-type genes. Hence, the somatic-type 5S RNA genes have a competitive advantage in binding the transcription factor TFIIIA in somatic cells and are thereby expressed to the exclusion of the oocyte-type genes. To test the replication-expression model, we determined the order of replication of the oocyte- and somatic-type 5S RNA genes. Xenopus cells were labeled with bromodeoxyuridine, stained for DNA content, and then sorted into fractions of S phase by using a fluorescence-activated cell sorter. The newly replicated DNA containing bromodeoxyuridine was separated from the lighter, unreplicated DNA by equilibrium centrifugation and was hybridized with DNA probes specific for the oocyte- and somatic-type 5S RNA genes. In this way we found that the somatic-type 5S RNA genes replicate early in S phase, whereas the oocyte-type 5S RNA genes replicate late in S phase, demonstrating a key aspect of the replication-expression model.

Site-directed mutagenesis with Escherichia coli DNA polymerase III holoenzyme.

Escherichia coli DNA polymerase III holoenzyme was used to synthesize double-stranded DNA from M13 single-stranded DNA hybridized to a phosphorylated synthetic oligodeoxynucleotide containing a nucleotide substitution. The resulting DNA was transfected into E. coli JM101 without further treatment. Sequence analysis of randomly chosen phage clones revealed that the efficiency of mutagenesis was nearly 50%, which is the theoretical maximum. Treatment with DNA ligase after DNA synthesis was not necessary to obtain high efficiency of mutagenesis. Thus, use of DNA polymerase III holoenzyme provides a simple and efficient procedure for site-directed mutagenesis.

Transcription of Xenopus 5S ribosomal RNA genes.

The accurate transcription of 5S RNA genes when injected into the nucleus of Xenopus oocytes or when added to an in vitro transcription system has allowed identification of the DNA sequences and one of the protein factors required for 5S RNA synthesis. Moreover, 5S RNA genes as part of intact chromosomes maintain a transcriptionally regulated state when injected into Xenopus oocyte nuclei. A detailed picture of the developmental regulation of 5S RNA gene expression is now emerging.

A comprehensive sequence analysis program for the IBM personal computer.

We have developed a versatile program for the analysis of nucleic acid and protein sequences on the IBM Personal Computer. The program is interactive and self-instructing. It contains all the features generally found in sequence analysis programs on large computers, including extensive homology routines, as well as new procedures for the entry of sequence data. The program contains facilities to store and utilize the entire Nucleic Acid Sequence Data Bank. We have devised a new algorithm to find restriction enzyme sites, which allows our microcomputer program to find all sites on a small plasmid for 100 different enzymes in 1 to 2 minutes.

Upstream sequences required for transcription of the TFIIIA gene in Xenopus oocytes.

The DNA sequences required for efficient initiation of transcription of the Xenopus transcription factor IIIA (TFIIIA) gene were determined by microinjecting a series of deletion and linker substitution mutants into Xenopus oocyte nuclei. An upstream activating sequence, which resides between residues -283 and -238, and perhaps a second sequence between -167 and -122 preceding the transcription start site, together stimulate transcription about 30-fold. The distance between the two sequences and the distance from them to the initiation site can vary by at least 13 base pairs without loss of activity. The TFIIIA upstream sequences can stimulate transcription of other genes, for example, they stimulate transcription from the herpes thymidine kinase promoter about 30-fold.

The reactivation of developmentally inert 5S genes in somatic nuclei injected into Xenopus oocytes.

In the frog Xenopus, the somatic-type 5S ribosomal RNA genes are active in all cells; the oocyte-type 5S genes are active in oocytes but not in somatic cells. A new method of native gel electrophoresis resolves the two types of 5S RNA which are of the same length but different sequence. When somatic nuclei were injected into oocytes, their inactive oocyte-type 5S genes often remained inactive, and had thus conserved their developmentally regulated condition. Other conditions, which include treatment with 0.35 M NaCl, reactivated the genes.

Analysis of biological sequences on small computers.

We review a wide range of programs for micro- and minicomputers that facilitate the collection and analysis of DNA, RNA, or protein sequences. Special-purpose programs that perform a single function and general-purpose programs that perform a set of functions are both considered. The information presented should be useful to any biologist who wishes to obtain a computer program to aid in sequence investigations.

Nucleotide sequence of Xenopus borealis oocyte 5S DNA: comparison of sequences that flank several related eucaryotic genes.

Genomic Xenopus borealis oocyte-specific 5S DNA (Xbo) contains clusters of 5S rRNA genes. The number of genes varies among clusters, and the distance between genes within a cluster is about 80 nucleotides. The spacer DNA between gene clusters is AT-rich and heterogeneous in length due in part to variable numbers of a tandemly repeated 21 nucleotide sequence. A cloned fragment of Xbo 5S DNA (Xbo1) containing three 5S rRNA genes has been sequenced. The sequences of Xbo1 genes 1 and 2 are very similar to the dominant 5S RNA sequence, whereas 15 of the 120 residues in the third gene are different. The sequence of gene 3 is as different from the dominant gene sequence as the X. laevis pseudogene is from the 5S RNA gene. Sequence analysis of genomic DNA shows that gene 3 is an abundant component of the multigene family. All three genes are transcribed when added to an extract of X. laevis oocyte nuclei, and a fragment of Xbo1 lacking the AT-rich spacer DNA and the 5' end of the first gene supports transcription of genes 2 and 3 in this in vitro system. Thus the 80 nucleotides preceding each 5S gene are sufficient for promoter function. Nucleic acid sequences preceding several eucaryotic genes that are transcribed by RNA polymerase III were analyzed and the following common features were found: a purine-rich region; at least one direct repeat; the absence of dyad symmetry; transcription beginning with a purine; a pyrimidine residue immediately preceding the first nucleotide of the gene; and the oligonucleotides AAAAG, AGAAG and GAC, located approximately 15, 25 and 35 nucleotides, respectively, before the start of transcription. The 10 base pair (bp) spacing between the homologous oligonucleotides is that expected for a recognition signal on one face of a DNA double helix. The extensive sequence differences between most of the spacers that precedes these genes make the three conserved oligonucleotides more striking. Parts of the 5' flanking regions of the three Xbo1 gene (-12 to -40), which include the conserved oligonucleotides, are identical. In contrast, 7 of the first 11 nucleotides that precede the third 5S RNA gene in Xbo1 differ from those that precede the first gene. The sequences following the X. borealis oocyte and somatic 5S genes are identical in 12 of the first 14 residues and contain two or more T clusters, as does the corresponding region of X. laevis oocyte 5S DNA. The 3' sequences of the Xenopus 5S rRNA genes and several other eucaryotic genes contain features in common with procaryotic transcription termination sites. The 3' end of the gene is GC-rich and contains a dyad symmetry. Termination occurs in an AT-rich region containing one or more T clusters on the noncoding strand.

Improvements to a program for DNA analysis: a procedure to find homologies among many sequences.

We have devised an algorithm for finding partial homologies among a set of nucleotide sequences. The algorithm and other improvements have been incorporated into a commonly used computer program for the analysis of sequence data.

Chromosomal mapping of Xenopus 5S genes: somatic-type versus oocyte-type.

Xenopus 5S RNA genes exhibit a pattern of differential expression during development in which some members (oocyte-type) are transcribed only in oocytes, while others (somatic-type) are expressed in both oocytes and somatic cells. Using cloned DNA probes specific for each gene type, we determined the positions of these genes on Xenopus metaphase chromosomes by in situ hybridization. Somatic-type 5S genes in both X. laevis and X. borealis are located at the distal end of the long arm of only one chromosome (number 9). The oocyte-type 5S RNA genes are found at the distal ends of the long arms of most Xenopus chromosomes, including chromosome 9. Thus, large scale differences in chromosomal location cannot explain the selective expression of these genes, as suggested previously.

Regulation of differential processing of mouse immunoglobulin mu heavy-chain mRNA.

The switch between the synthesis of membrane-bound and secreted IgM during B cell differentiation is accomplished by producing, from a single gene, two alternative forms of mu heavy-chain mRNA that differ only in their 3' termini. The precursor mu RNA is either polyadenylated at the first poly(A) site, for secreted mu mRNA, or spliced between the C4 and M1 exons, for membrane-bound mu mRNA, in a mutually exclusive manner. To elucidate the molecular mechanism of the differential processing of mouse mu mRNA, we analyzed the expression of various mouse mu gene constructs stably transfected into mouse cell lines. In B cell lines, processing of the exogenously transfected mu gene transcripts accurately reflected the developmental stage of the recipient cells: both secreted and membrane-bound mu mRNAs are produced in early-stage B cells while secreted mu mRNA is primarily produced in late-stage B cells. In fibroblast cell lines, mu mRNAs transcribed from the Moloney murine sarcoma virus LTR promoter were processed primarily to the secreted form. Thus, production of the secreted form seems to be the non-regulated processing pattern. When the splicing signal of the C4-M1 intron was mutagenized, polyadenylation at the first poly(A) site occurred efficiently regardless of the recipient cell lines. On the other hand, when the polyadenylation signal was mutagenized, the splicing occurred efficiently in early-stage B cells, but only weakly in late-stage B cells and fibroblast cells. These results suggest that the splicing of the C4-M1 intron is stimulated in early-stage B cells.

Transcription initiation of Xenopus 5S ribosomal RNA genes in vitro.

We have studied initiation of transcription of 5S RNA genes in extracts of Xenopus laevis oocyte nuclei. To aid in this study we developed a general assay for specificity of transcription initiation that does not require accurate termination of transcription. Following in vitro transcription with gamma-32P-labeled nucleoside triphosphages, the RNA is digested with pancreatic RNAase and fingerprinted by two dimensional chromatography. A 5S RNA gene with a variant sequence, in which the G residue at position +1 is replaced by a C, initiates transcription at an A residue one nucleotide preceding the C. Although Xenopus RNA polymerase form III can initiate transcription at many sites on plasmid DNA, all of the transcripts start with purines. The majority of these purines are triphosphorylated. When a repeating unit of Xenopus 5s DNA is inserted into the plasmid, initiations at the vector start sites are suppressed and the major labeled 5' oligonucleotide is derived from 5S RNA.

Computer analysis of nucleic acid regulatory sequences.

We describe a computer program designed to facilitate the analysis of nucleic acid sequences. The program can search several nucleic acid sequences for oligonucleotides common to all of them. It can examine a DNA or RNA sequence for two kinds of homologous regions--repetitions and dyad symmetries. The homologies need not be perfect: mismatches and "looping out" of nucleotides are allowed. The program also finds (A+T)- and (G+C)-rich regions, locates restriction enzyme recognition sites, determines the distribution of di- and trinucleotides, and performs various other functions. We include two representative applications of the program. All published prokaryotic transcription termination sequences (June 1977) were found to share the following features: (i) a string of at least five T residues, (ii) the sequence CGGGC or a close analog immediately preceding the T cluster, (iii) a region of strong dyad symmetry preceding the Ts and including the CGGGC sequence. A sequence of 221 nucleotides consisting of the Escherichia coli trp promoter, operator, and leader was found to contain two strong dyad symmetries. These homologies both occur at known regulatory sites; no comparable homologies occur in regions without regulatory significance.