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Biomedical subjects

R Grosschedl

Publications and source records attributed to R Grosschedl.

At least 73 records · Page 4Linked to original sources
PubMed1991

LEF-1, a gene encoding a lymphoid-specific protein with an HMG domain, regulates T-cell receptor alpha enhancer function [corrected].

Lymphoid-specific cDNA clones were isolated that encode a nuclear protein with homology to the chromosomal nonhistone protein HMG-1 and to putative regulators of cell specialization, including the mammalian testis-determining factor SRY and fungal mating-type proteins. The gene represented by the isolated cDNA clones, termed LEF-1 (lymphoid enhancer-binding factor 1), is developmentally regulated and expressed in pre-B and T lymphocytes but not in later-stage B cells or nonlymphoid tissues. Both endogenous and recombinant LEF-1 were shown to bind to a functionally important site in the T-cell antigen receptor (TCR) alpha enhancer. Maximal TCR alpha enhancer activity was found to parallel the cell type-specific expression pattern of LEF-1. Moreover, forced expression of recombinant LEF-1 in late stage B cells increases TCR alpha enhancer function. Taken together, these data suggest that LEF-1 is a regulatory participant in lymphocyte gene expression and differentiation.

Amino Acid Sequence↗
PubMed1991

Complex pattern of immunoglobulin mu gene expression in normal and transgenic mice: nonoverlapping regulatory sequences govern distinct tissue specificities.

Analysis of normal mice and transgenic mice carrying a rearranged immunoglobulin mu gene revealed a more complex tissue-specific pattern of mu gene expression than anticipated from previous observations. Expression of the endogenous mu locus and the mu transgene was detected both in lymphoid tissues and in skeletal muscle. Analysis of the expression pattern of mu transgenes containing intragenic deletions or point mutations in binding sites for Oct transcription factors (OCTA sites) indicated that distinct regulatory sequences control lymphoid- and muscle-specific mu gene expression. Consistent with previous transfection experiments, mu gene expression in lymphoid tissues is dependent on the intragenic enhancer and the OCTA site in the promoter. However, neither of these regulatory sequences is required for mu gene expression in skeletal muscle that is governed by a muscle-specific control region located 3' of the enhancer. An "off-state" of the mu transgene was observed only in liver and embryonal fibroblasts, whereas enhancer-dependent mu transgene expression was detected at low levels in other nonlymphoid tissues. From these data we suggest a model for the regulation of tissue-specific mu gene expression in which a "ubiquitous" competence for basal transcription is up-regulated in lymphoid and muscle tissues by distinct cell type-specific regulatory sequences and down-regulated in liver and fibroblastic cells by putative negative sequence elements.

Animals↗
PubMed1991

Heterogeneously initiated transcription from the pre-B- and B-cell-specific mb-1 promoter: analysis of the requirement for upstream factor-binding sites and initiation site sequences.

The mb-1 gene, encoding a membrane immunoglobulin-associated protein, is developmentally regulated and expressed specifically in pre-B and mature B lymphocytes. Analysis of the TATA-less mb-1 promoter indicated that it directs initiation of transcription from multiple sites. Promoter sequences between -68 and +70 conferred the correct pattern of cell type-specific transcription upon a heterologous gene. Two nuclear factor-binding sites that are important for promoter function were identified between -59 and -38. Both sites interacted with ubiquitous nuclear factors in vitro. One of these factors was identified as Sp1. Multimerized copies of both factor-binding sites augmented expression from a heterologous minimal promoter in both lymphoid and nonlymphoid cells, suggesting that additional mb-1 promoter sequences are involved in determining the correct cell type specificity. Analysis of the heterogeneity of transcription initiation indicated that a mutation which increased the distance between upstream sequences and the region of initiation resulted in the utilization of a novel set of initiation sites. Moreover, an insertion of a TATA element into the mb-1 promoter at -30 biased initiation of transcription to +1 but did not abolish the use of the other sites. Mutation of an initiator sequence homology encompassing one of the major initiation sites had only a minor effect on its utilization. From these data, we conclude that upstream factor-binding sites in the TATA-less mb-1 promoter define a region in which initiation of transcription occurs at multiple sites.

Animals↗
PubMed1988

Expression of the int-1 gene in transgenic mice is associated with mammary gland hyperplasia and adenocarcinomas in male and female mice.

Transcriptional activation of the int-1 gene by proviral insertion mutations is thought to be a key step in mammary tumor induction by the mouse mammary tumor virus (MMTV). To test this hypothesis, we have constructed an int-1 allele resembling those found in virus-induced tumors, with an MMTV LTR placed 5' to the int-1 gene in the opposite transcriptional orientation. Transgenic mice harboring this allele express int-1 RNA at high levels in mammary and salivary glands of male and female mice and in male reproductive organs. The mammary glands of males and virgin females are grossly hyperplastic compared with those of nontrasgenic littermates. Mammary and (less frequently) salivary adenocarcinomas occur in these animals at rates indicating that transcriptional activation of int-1 and associated hyperplasia are initiating events in multistep carcinogenesis.

Adenocarcinoma↗
PubMed1988

Stable propagation of the active transcriptional state of an immunoglobulin mu gene requires continuous enhancer function.

Enhancer sequences control the expression of the immunoglobulin mu heavy chain gene. In this study we examined whether the active transcriptional state of the mu gene can be propagated from mother to daughter cells after deletion of its enhancer. A mu gene that contains the enhancer flanked by D and J recombination signals was stably introduced into pre-B cells, resulting in mu gene transcription. Subsequent deletion of the enhancer from the transfected gene by D-to-J joining reproducibly resulted in switching off of mu expression. Moreover, transcriptional inactivation occurred although the mu gene was found to be hypomethylated prior to deletion of the enhancer. We conclude that the enhancer does not confer upon the mu gene "memory" of its active transcriptional state. Thus, the enhancer seems to be required both for establishment and for maintenance of mu gene expression.

Base Sequence↗
PubMed1985

Cell-type specificity of immunoglobulin gene expression is regulated by at least three DNA sequence elements.

The regulation of cell-type specificity of immunoglobulin (lg) mu heavy chain (H) gene expression was examined by introducing various hybrid genes containing lg gene sequences combined with portions of a tissue-nonspecific transcription unit into lymphoid and nonlymphoid cells. Replacing the lymphocyte-specific IgH enhancer with a viral enhancer did not affect tissue specificity of mu Ig gene expression. We identified two new regulatory regions that provide transcriptional tissue specificity. First, the V H promoter region between position -154 and +57 was shown to direct lymphocyte-specific transcription of a bacterial gpt gene, even in the presence of a viral enhancer. Second, mu intragenic sequences, lacking the IgH enhancer, were found to regulate the level of accumulated Ig transcripts in a tissue-specific fashion. These results demonstrate that tissue specificity of Ig gene expression is not solely regulated by the enhancer but that the promoter, and as yet undefined intragenic sequences, contain lymphoid-specific regulatory information.

Animals↗
PubMed1984

Introduction of a mu immunoglobulin gene into the mouse germ line: specific expression in lymphoid cells and synthesis of functional antibody.

A functionally rearranged mu heavy chain immunoglobulin (lg) gene was introduced into the germ line of mice. The mu gene encodes a polypeptide which, combined with lambda 1 light chains, shows a specificity for binding the hapten NP. Four transgenic mice harboring 20-140 copies of the foreign mu gene expressed the gene specifically in spleen, lymph node, and thymus at a high level. Purified surface lg-positive B cells, Lyt 2-positive mature T cells, and thymocytes transcribed the foreign mu gene at a similarly high level, suggesting that control of lg gene rearrangement might be the only mechanism that determines the specificity of heavy chain gene expression within the lymphoid cell lineage. No transcription of the foreign mu gene was detected in nonlymphoid tissues with the exception of the heart which expressed the gene at a low level. The transgenic mice had up to 400-fold elevated serum levels of NP binding antibody, which contained a heavy chain with the characteristics of the foreign mu gene. The serum levels of endogenous heavy and light chains in transgenic mice appeared to be the same as in normal mice.

Animals↗
PubMed1984

Two regulatory elements for immunoglobulin kappa light chain gene expression.

By using internal deletions within a rearranged immunoglobulin kappa light chain gene, the presence of an intron regulatory sequence (enhancer) has been confirmed. Its presence is required for high-level transcription from a plasmid after transfection into myeloma cells. Transfection efficiency was monitored by the activity of a deleted H4 histone gene included in the plasmid. The intron element could be moved upstream of the gene in both orientations, fulfilling the definition of an enhancer. By using 5' deletions, a second regulatory element was located upstream of the "TATA" box, between positions -69 and -104. These two elements both are required for efficient kappa chain gene expression.

Animals↗
PubMed1983

A functional component of the sea urchin H2A gene modulator contains an extended sequence homology to a viral enhancer.

The DNA sequences imparting a maximal rate of sea urchin H2A gene transcription in the frog oocyte nucleus were narrowed down by deletion mapping to a DNA segment -165 to -111, far-upstream of the H2A mRNA cap site. C to T base changes in this area create strong down mutations, hence the primary structure of this DNA sequence is of paramount importance to the H2A gene expression. Sequence comparisons suggest that the -165 to -111 region may contain two essential sequence blocks. Most strikingly, the -135 area contains a 14 out of 17 basepair homology to the Moloney murine sarcoma virus enhancer and to topologically related 5' LTR-sequences of the simian sarcoma virus and the murine Friend spleen focus forming virus.

Animals↗
PubMed1982

Generation of authentic 3' termini of an H2A mRNA in vivo is dependent on a short inverted DNA repeat and on spacer sequences.

We have determined what sequences are required to generate the authentic 3' termini of a sea urchin H2A histone mRNA. We have constructed a series of deletion and insertion mutants in the cloned histone repeat unit h22 of Psammechinus miliaris and have analyzed the transcripts of both wild-type and mutant DNAs produced in the frog oocyte. The protein-coding sequences of the H2A gene can be removed without any deleterious effects on transcription initiation or termination. A 12 bp deletion, which removes a highly conserved inverted DNA repeat immediately preceding the H2A mRNA 3' terminus, elicits read-through of the polymerase into the spacer DNA further downstream. However, the inverted repeat and the sequence coding for the 3' terminus of the mRNA are by themselves not sufficient to generate faithful 3' ends. Our data suggest that spacer sequences downstream of the 3' mRNA terminus are required as well.

Animals↗
PubMed1982

Delimitation of far upstream sequences required for maximal in vitro transcription of an H2A histone gene.

Sea urchin (psammechinus miliaris) H2A histone genes shown to be promoter mutants from oocyte injection experiments were tested for their ability to initiate transcription in vitro. Circular templates were transcribed with HeLa cell extracts, and the transcripts were assayed by mung bean or S1 nuclease mapping of the 5' ends. The transcripts of the H2A mutants produced in vitro were qualitatively similar and, in most cases, identical to those seen in oocyte injection experiments, but quite large quantitative differences were observed for some H2A mutant genes. Both the T-A-T-A box and far upstream sequences residing in the modulator segment E [Grosschedl, R. & Birnstiel, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 7102--7106] were found to be essential for maximal transcription in vitro. Deletion of either of these sequence elements reduced transcription to 20%. A similar reduction in the amount of H2a transcripts was found when a T-A-T-A-to-T-A-G-A point mutant was tested in vitro. Essential far upstream sequences were mapped between nucleotides -139 and -111, 5' to the initiation site of transcription. In the standard run-off transcription test using restriction fragments, the effects of these sequences could be mimicked by free DNA ends, suggesting that the function of this in vitro upstream sequence might be to provide an entry side for RNA polymerase II.

Base Sequence↗
PubMed1981

ClaI. a new restriction endonuclease from Caryophanon latum L.

From Caryophanon latum L site specific restriction endonuclease (ClaI) has been purified, which recognises tha DNA hexanucleotide palindrome 5'-A-T-C-G-A-T-3'. Staggered cleavage generates DNA restriction fragments with 5'-terminal pCG extensions. A CLaI map of bacteriophage lambda has been determined, which indicates cleavage inhibition due to adenine methylation at over lapping ClaI-GATC recognition sequences. Plasmid pBR322 is cut only once, in the tetracycline promoter region, and can, therefore, be used as a vector system for cloning fragments derived from ClaI digestions, and in addition for fragments generated by TaqI, HpaII, and several other enzymes.

Bacteriophage lambda↗
PubMed1980

Spacer DNA sequences upstream of the T-A-T-A-A-A-T-A sequence are essential for promotion of H2A histone gene transcription in vivo.

The control region of a sea urchin H2A histone gene may be functionally dissected into at least three DNA segments, which we have termed modulator, selector, and initiator elements. While the initiator and in particular the selector containing the T-A-T-A-A-A-T-A sequence are specificity elements that dictate the generation of faithful 5' ends to H2A mRNA, the modulators control the rate at which these specificity elements operate [Grosschedl, R. & Birnstiel, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 1432-1436]. By functional tests of in vitro mutated histone DNA in the Xenopus oocyte we have now discovered that the segment E of the A+T-rich spacer DNA lying at a considerable distance upstream of the conservative T-A-T-A-A-A-T-A sequence is a strong modulator element of H2A gene transcription. Deletion of this element creates a 15- to 20-fold H2A-specific down mutation. Segment E by itself cannot elicit initiation of transcription except in coordination with the prelude sequence of the H2A gene. The nucleotide sequence of the relevant spacer element showing modulator activity has been determined and found to contain a pattern of T and A runs as well as a series of inverted repeats. Additional pre-H2A spacer mutants, including a spacer inversion mutant, have been constructed in vitro, that, when injected into the oocyte nucleus, modulate the expression of the H2A gene by an overall factor as large as 100. Other factors controlling promoter activity are discussed.

Animals↗
PubMed1980

Identification of regulatory sequences in the prelude sequences of an H2A histone gene by the study of specific deletion mutants in vivo.

Conserved DNA sequence elements of putative regulatory functions were deleted from the prelude region of a sea urchin H2A histone gene. For this, the wild-type H2A gene of the 6-kilobase histone DNA repeat unit was replaced by various mutant H2A genes by cloning. The effects of the manipulation on H2A mRNA synthesis were studied by injection of the mutant DNAs into centrifuged Xenopus oocytes. The unmanipulated H2B gene residing within the same repeat unit provided a suitable internal control for these studies. Deletion of the T-A-T-A-A-A-T-A motif, once thought to be the functional equivalent of the bacterial Pribnow box, did not abolish transcription of the gene; instead, a number of novel mRNA 5' termini were generated. We argue that the T-A-T-A-A-A-T-A motif is a specificity element, a selector of eukaryotic gene transcription. Deletion of the "cap-sequence," 5' pyrimidine-C-A-T-T-C-purine 3' and most of the mRNA leader sequence did not abolish transcription but created yet another mRNA 5' terminus. In contrast to these deletions, which are both down-mutations, deletion of H2A gene-specific conserved DNA sequences upstream from the T-A-T-A-A-A-T-A motif enhanced mRNA synthesis. A hypothesis for the function of these DNA sequences as eukaryotic promoter elements is discussed.

Animals↗
PubMed1979

A tRNA gene of Xenopus laevis contains at least two sites promoting transcription.

A small cloned DNA segment previously shown to contain all genetic information for the expression of the tRNA1met gene of Xenopus laevis was cleaved into an anterior and posterior portion by Hae III restriction. Both restriction fragments were cloned in pCR1 using EcoRI linkers. Starting from these tDNA subclones, a series of new recombinants were constructed. The transcriptional activity of the cloned DNA's was tested both in an in vitro transcriptional system and by means of the oocyte injection technique. It was shown that both the 5' and 3' ends of the cloned gene unit were essential for transcription. We have developed a model for the functional organization of the tRNA1met gene. We propose that the gene contains a regulatory site situated near the 3' portion of the gene unit. For transcription to occur both in vivo and in vitro a specific initiation site near the 5' end of the gene is required.

Animals↗
PubMed1979

DNA sequences and structural homologies of the replication origins of lambdoid bacteriophages.

The DNA sequences for the origins of replication of the lambdoid bacteriophages phi80, 434, phi21, and lambdaimm21 (identical to phi21) have been determined and compared to the lambda structure. Two presumptive elaborate binding sites for two initiator proteins have been identified in their outer sections, while a replicational primer start site seems to be located in their centres.

Base Sequence↗