CD4+ T cells reactive to enteric bacterial antigens in spontaneously colitic C3H/HeJBir mice: increased T helper cell type 1 response and ability to transfer disease.

C3H/HeJBir mice are a new substrain that spontaneously develop colitis early in life. This study was done to determine the T cell reactivity of C3H/HeJBir mice to candidate antigens that might be involved in their disease. C3H/HeJBir CD4+ T cells were strongly reactive to antigens of the enteric bacterial flora, but not to epithelial or food antigens. The stimulatory material in the enteric bacteria was trypsin sensitive and restricted by class II major histocompatibility complex molecules, but did not have the properties of a superantigen. The precursor frequency of interleuken (IL)-2-producing, bacterial-reactive CD4+ T cells in colitic mice was 1 out of 2,000 compared to 1 out of 20,000-25,000 in noncolitic control mice. These T cells produced predominately IL-2 and interferon gamma, consistent with a T helper type 1 cell response and were present at 3-4 wk, the age of onset of the colitis. Adoptive transfer of bacterial-antigen-activated CD4+ T cells from colitic C3H/HeJBir but not from control C3H/HeJ mice into C3H/HeSnJ scid/scid recipients induced colitis. These data represent a direct demonstration that T cells reactive with conventional antigens of the enteric bacterial flora can mediate chronic inflammatory bowel disease.

Epithelial cell differentiation in normal and transgenic mouse intestinal isografts.

Transgenes consisting of segments of the rat liver fatty acid-binding protein (L-FABP) gene's 5' non-transcribed domain linked to the human growth hormone (hGH) gene (minus its regulatory elements) have provided useful tools for analyzing the mechanisms that regulate cellular and spatial differentiation of the continuously renewing gut epithelium. We have removed the jejunum from normal and transgenic fetal mice before or coincident with, cytodifferentiation of its epithelium. These segments were implanted into the subcutaneous tissues of young adult CBY/B6 nude mouse hosts to determine whether the bipolar, migration-dependent differentiation pathways of gut epithelial cells can be established and maintained in the absence of its normal luminal environment. Immunocytochemical analysis of isografts harvested 4-6 wk after implantation revealed that activation of the intact endogenous mouse L-FABP gene (fabpl) in differentiating enterocytes is perfectly recapitulated as these cells are translocated along the crypt-to-villus axis. Similarly, Paneth and goblet cells appear to appropriately differentiate as they migrate to the crypt base and villus tip, respectively. The enteroendocrine cell subpopulations present in intact 4-6-wk-old jejunum are represented in these isografts. Their precise spatial distribution along the crypt-to-villus axis mimics that seen in the intact gut. A number of complex interrelationships between enteroendocrine subpopulations are also recapitulated. In both "intact" and isografted jejunum, nucleotides -596 to +21 of the rat L-FABP gene were sufficient to direct efficient expression of the hGH reporter to enterocytes although precocious expression of the transgene occurred in cells located in the upper crypt, before their translocation to the villus base. Inappropriate expression of hGH occurred in a high percentage (greater than 80%) of secretin, gastrin, cholecystokinin, and gastric inhibitory peptide producing enteroendocrine cells present in the intact jejunum of 4-6-wk-old L-FABP-596 to +21/hGH transgenics. Addition of nucleotides -597 to -4,000 reduced the percentage of cells co-expressing this reporter four- to eightfold in several of the subpopulations. Jejunal isografts from each transgenic pedigree studied contained a lower percentage of hGH positive enteroendocrine cells than in the comparably aged intact jejunum.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of transgenic mice to map cis-acting elements in the intestinal fatty acid binding protein gene (Fabpi) that control its cell lineage-specific and regional patterns of expression along the duodenal-colonic and crypt-villus axes of the gut epithelium.

The mouse intestinal epithelium is able to establish and maintain complex lineage-specific, spatial, and temporal patterns of gene expression despite its rapid and continuous renewal. A multipotent stem cell located near the base of each intestinal crypt gives rise to progeny which undergo amplification and allocation to either enterocytic, Paneth cell, goblet cell, or enteroendocrine cell lineages. Differentiation of these four lineages occurs during their geographically ordered migration along the crypt-villus axis. Gut stem cells appear to have a "positional address" which is manifested by differences in the differentiation programs of their lineal descendants along the duodenal-colonic (cephalocaudal) axis. We have used the intestinal fatty acid binding protein gene (Fabpi) as a model to identify cis-acting elements which regulate cell- and region-specific patterns of gene expression in the gut. Nucleotides -1178 to +28 of rat Fabpi direct a pattern of expression of a reporter (human growth hormone [hGH]) which mimics that of mouse Fabpi (a) steady-state levels of hGH mRNA are highest in the distal jejunum of adult transgenic mice and fall progressively toward both the duodenum and the mid-colon; and (b) hGH is confined to the enterocytic lineage and first appears as postmitotic, differentiating cells exit the crypt and migrate to the base of small intestinal villi or their colonic homologs, the surface epithelial cuffs. Nucleotides -103 to +28, which are highly conserved in rat, mouse and human Fabpi, are able to correctly initiate transgene expression in late fetal life, restrict hGH to the enterocytic lineage, and establish an appropriate cephalocaudal gradient of reporter expression. This cephalocaudal gradient is also influenced by cis-acting elements located between nucleotides -1178 and -278, and -277 and -185 that enhance and suppress (respectively) expression in the ileum and colon and by element(s) located upstream of nucleotide -277 that are needed to sustain high levels of hGH production after weaning. Nucleotides -277 to -185 contain part of a domain conserved between the three orthologous Fabpi genes (nucleotides -240 to -159), a 24-bp element (nucleotides -212 to -188) that binds nuclear factors present in colonic but not small intestinal epithelial cells, and a portion of a CCAAT/enhancer binding protein footprint (C/EBP alpha, nucleotides -188 to -167). Removal of nucleotides -277 to -185 (yielding I-FABP-184 to +28/hGH+3) results in inappropriate expression of hGH in proliferating and nonproliferating epithelial cells located in the mid and upper portions of duodenal, jejunal, ileal, and colonic crypts without affecting the "shape" of the cephalocaudal gradient of transgene expression.(ABSTRACT TRUNCATED AT 400 WORDS)

Murine mucopolysaccharidosis type VII: long term therapeutic effects of enzyme replacement and enzyme replacement followed by bone marrow transplantation.

We demonstrated previously that short term administration of recombinant beta-glucuronidase to newborn mice with mucopolysaccharidosis type VII reduced lysosomal storage in many tissues. Lysosomal storage accumulated gradually after cessation of enzyme replacement therapy. Mice alive at 1 yr of age had decreased bone deformities and less lysosomal storage in cortical neurons. Here we compare the effects of long term enzyme replacement initiated either at birth or at 6 wk of age, and of enzyme administration initiated at birth followed by syngeneic bone marrow transplantation (BMT) at 5 wk of age. Several mice from each treatment group lived to at least 1 yr of age. Liver and spleen samples had beta-glucuronidase levels ranging from 2.4 to 19.8% of normal and showed a parallel decrease in lysosomal storage. The combination of enzyme replacement therapy followed by BMT reduced lysosomal distension in meninges, corneal fibroblasts, and bone when compared with treatment with enzyme alone. Mice treated at birth had less lysosomal storage in some neurons of the brain and the skeletal dysplasia was less severe when compared to mice whose treatment was delayed until 6 wk of age. We conclude that both enzyme replacement alone and early enzyme replacement followed by BMT have long term positive effects on murine mucopolysaccharidosis type VII. In addition, treatment started at birth is far more effective than treatment initiated in young adults.

Murine mucopolysaccharidosis type VII. Characterization of a mouse with beta-glucuronidase deficiency.

We have characterized a new mutant mouse that has virtually no beta-glucuronidase activity. This biochemical defect causes a murine lysosomal storage disease that has many interesting similarities to human mucopolysaccharidosis type VII (MPS VII; Sly syndrome; beta-glucuronidase deficiency). Genetic analysis showed that the mutation is inherited as an autosomal recessive that maps to the beta-glucuronidase gene complex, [Gus], on the distal end of chromosome 5. Although there is a greater than 200-fold reduction in the beta-glucuronidase mRNA concentration in mutant tissues, Southern blot analysis failed to detect any abnormalities in the structural gene, Gus-sb, or in 17 kb of 5' flanking and 4 kb of 3' flanking sequences. Surprisingly, a sensitive S1 nuclease assay indicated that the relative level of kidney gusmps mRNA responded normally to androgen induction by increasing approximately 11-fold. Analysis of this mutant mouse may offer valuable information on the pathogenesis of human MPS VII and provide a useful system in which to study bone marrow transplantation and gene transfer methods of therapy.

Enzyme replacement therapy for murine mucopolysaccharidosis type VII.

Recombinant mouse beta-glucuronidase administered intravenously to newborn mice with mucopolysaccharidosis type VII (MPS VII) is rapidly cleared from the circulation and localized in many tissues. Here we determine the tissue distribution of injected enzyme and describe its effects on the histopathology in 6-wk-old MPS VII mice that received either one injection of 28,000 U recombinant beta-glucuronidase at 5 wk of age or received six injections of 28,000 U given at weekly intervals beginning at birth. These mice were compared with untreated 6-wk-old MPS VII mice. The single injection decreased lysosomal distention in the fixed tissue macrophage system. MPS VII mice that received multiple injections had 27.8, 3.5, and 3.3% of normal levels of beta-glucuronidase in liver, spleen, and kidney, respectively. Brain had detectable beta-glucuronidase, ranging from 2.0-12.1% of normal. Secondary elevations of alpha-galactosidase and beta-hexosaminidase in brain, spleen, liver, and kidney were decreased compared with untreated MPS VII mice. Although no improvement was observed in chondrocytes, glia, and some neurons, the skeleton had less clinical and pathological evidence of disease and the brain had reduced lysosomal storage in meninges and selected neuronal groups. These data show that recombinant beta-glucuronidase treatment begun in newborn MPS VII mice provides enzyme to most tissues and significantly reduces or prevents the accumulation of lysosomal storage during the first 6 wk of life. Whether therapy begun later in life can achieve this level of correction remains to be established.

Sequence conservation and structural organization of the glycerol-3-phosphate dehydrogenase promoter in mice and humans.

Cloned segments of the mouse glycerol-3-phosphate dehydrogenase (GPDH) gene, Gdc-1, were used to screen a human library. Human clones obtained spanned 25 kilobases of genomic DNA containing the human GPDH gene, GPD1. The 4 kb of sequence obtained from the 5'-flanking region and first exon of GPD1 was compared with the corresponding mouse sequence. Both sequences share a HindIII site located in what has proven to be the highly conserved 3' untranslated region of an upstream gene of unknown function, D15Kzl. The 3.6-kilobase segment of mouse DNA located between D15Kzl and Gdc-1 was provisionally termed the GPDH promoter. Alignment of the mouse promoter with the corresponding human sequence revealed two conserved domains. An upstream distal promoter region is approximately 900 base pairs in length. A downstream or proximal promoter region consists of approximately 300 base pairs immediately upstream of a TATA-like box and contains the fat-specific elements 1 and 2. Analysis of the chromatin structure of the Gdc-1 promoter revealed four DNase I-hypersensitive sites. They were present in DNA of liver and brown fat, in which GPDH expression is high, but were absent in DNA of spleen, in which GPDH expression is low. Methylation studies of the promoter showed it to be heavily methylated in sperm. However, the DNA from each adult somatic tissue had a unique distribution of nonmethylated sites and could easily be identified by its methylation pattern. These data suggest a structural model of the promoter that explains how Gdc-1 expression is differentially regulated in many types of cells.

Intracisternal A-particle element transposition into the murine beta-glucuronidase gene correlates with loss of enzyme activity: a new model for beta-glucuronidase deficiency in the C3H mouse.

The severity of human mucopolysaccharidosis type VII (MPS VII), or Sly syndrome, depends on the relative activity of the enzyme beta-glucuronidase. Loss of beta-glucuronidase activity can cause hydrops fetalis, with in utero or postnatal death of the patient. In this report, we show that beta-glucuronidase activity is not detectable by a standard fluorometric assay in C3H/HeOuJ (C3H) mice homozygous for a new mutation, gusmps2J. These gusmps2J/gusmps2J mice are born and survive much longer than the previously characterized beta-glucuronidase-null B6.C-H-2(bm1)/ByBir-gusmps (gusmps/gusmps) mice. Northern blot analysis of liver from gusmps2J/gusmps2J mice demonstrates a 750-bp reduction in size of beta-glucuronidase mRNA. A 5.4-kb insertion in the Gus-sh nucleotide sequence from these mice was localized by Southern blot analysis to intron 8. The ends of the inserted sequences were cloned by inverse PCR and revealed an intracisternal A-particle (IAP) element inserted near the 3' end of the intron. The sequence of the long terminal repeat (LTR) regions of the IAP most closely matches that of a composite LTR found in transposed IAPs previously identified in the C3H strain. The inserted IAP may contribute to diminished beta-glucuronidase activity either by interfering with transcription or by destabilizing the message. The resulting phenotype is much less severe than that previously described in the gusmps/gusmps mouse and provides an opportunity to study MPS VII on a genetic background that clearly modulates disease severity.

Spx1, a novel X-linked homeobox gene expressed during spermatogenesis.

Spx1, a novel mouse homeobox gene, encodes a homeodomain characteristic of the paired-like class of homeobox genes and has been mapped to the distal end of the X chromosome. Northern blot hybridization of adult tissues detected high levels of a single Spx1 transcript in the testis. Further analysis by in situ hybridization revealed predominant Spx1 expression within the spermatogonia/preleptotene spermatocytes and round spermatids of spermatogenic stages IV-VII. These expression data suggest SPX1 may play a role in the regulation of spermatogenesis.

Advantages of gradient vs. 5-fluorouracil enrichment of stem cells for retroviral-mediated gene transfer.

Retrovirally mediated gene transfer into murine totipotent hematopoietic stem cells (THSC) may be more efficient when the donor stem cells are enriched. We have used a rapid, nontoxic density gradient separation of mouse marrow to enrich stem cells. By characterizing the cell types in various fractions of the gradient, we found the majority of the THSC, spleen colony forming stem cells (CFU-S), erythroid burst forming cells (BFU-E) and dividing cells were in the same fraction. The gradient enrichment technique was then compared with one requiring 5-fluorouracil (5-FU) treatment of donor mice prior to marrow harvest. Cells enriched by both methods were tested for their ability to mediate retroviral gene transfer into normal mice. Gradient enrichment provided only one third as many nucleated cells as 5-FU treatment from the same number of donors. During the subsequent 4-day in vitro exposure to the retrovirus and growth factors, however, the number of gradient enriched cells increased 1.6-fold while the number of 5-FU treated cells decreased 3-fold. In lethally irradiated recipients, there was no difference between gradient and 5-FU enriched donor cells in the proportion of cells that generated CFU-S nor in the percentage of CFU-S that were infected. Secondary hosts did show differences. Gradient-enriched cells maintained more survivors for up to 6 months posttransplantation and more of the survivors were positive for the retrovirus. It is clear that the gradient method provides a rapid means to enrich CFU-S and THSC without exposure to the toxic effects of 5-FU.

Retroviral gene transfer into the intestinal epithelium.

The epithelial cells of the gastrointestinal tract may be attractive targets for somatic gene therapy. In these studies, we have used rats and mice to explore the feasibility of gene transfer into the small intestinal epithelium using retroviral vectors. The first series of experiments was conducted in mature Sprague-Dawley rats using an ecotropic retroviral vector that has bacterial beta-galactosidase (beta-Gal) as the reporter gene. The vector was introduced into the lumen of ligated segments of terminal ileum. After a 4-hr exposure period, the ligatures were removed. Sham-operated animals were subjected to the same ligation procedure but received only tissue culture medium in the ligated segment. All animals were sacrificed 6 days later, and tissue from both the experimental segment and an upstream control segment was assessed for cytoplasmic beta-Gal activity using X-Gal histochemistry. Expression of the reporter gene was observed in the crypt epithelium of tissue exposed to the vector. In the villus epithelium, high background staining precluded accurate assessment of reporter gene expression. To obviate the latter problem, we sought an alternative reporter gene for which there would be no background staining in control animals. We repeated the experiments with beta-glucuronidase as the reporter gene in MPS VII mutant mice, which are devoid of this enzyme. In these studies, ileal segments exposed to the vector demonstrated expression of the reporter gene in both the crypt and villus epithelium 4 days after exposure. These results indicate that genes can be transferred into the intestinal epithelium using retroviral vectors introduced luminally.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene therapy for murine mucopolysaccharidosis type VII.

Mucopolysaccharidosis type VII (MPS VII) is caused by a deficiency in the lysosomal enzyme beta-glucuronidase resulting in the accumulation of undegraded glycosaminoglycans in many tissues. A murine model of MPS VII shares many of the clinical, biochemical and histopathological features of human MPS VII and has provided an opportunity to study novel therapeutic approaches in a system with a uniform genetic background. Retroviral mediated gene therapy directed to the hematopoietic system or to artificial neo-organs resulted in low levels of enzyme in several tissues and reduced lysosomal storage in the liver and spleen. Partial correction of the disease in the eye was observed following an intravitreal injection of recombinant adenovirus. Neither retroviral nor adenoviral mediated gene transfer techniques resulted in a systemic reduction of lysosomal storage. Here we discuss several novel gene transfer approaches designed to increase the systemic levels of beta-glucuronidase in the MPS VII mouse.

A single-base-pair deletion in the beta-glucuronidase gene accounts for the phenotype of murine mucopolysaccharidosis type VII.

Murine mucopolysaccharidosis type VII is a heritable disease caused by a spontaneous mutation, gus(mps), closely linked to the beta-glucuronidase structural gene on chromosome 5. Mice homozygous for the mutation have a > 200-fold decrease in beta-glucuronidase mRNA levels and virtually no enzyme activity detectable by a sensitive fluorometric assay. Approximately 20 kb of genomic DNA containing the beta-glucuronidase gene Gus and > 2 kb of 5' and 3' flanking sequences were cloned from both a gus(mps)/gus(mps) mouse and a +/+ mouse of the progenitor strain. Restriction enzyme digests containing DNA fragments 20-400 bp in length were generated from each of the two Gus alleles and then compared by using nondenaturing polyacrylamide DNA-sequencing gels. This method rapidly identified a large number of restriction sites and was sensitive enough to detect a restriction fragment length variation resulting from a 1-bp deletion in the gus(mps) allele. DNA-sequence analysis of the mutant genomic fragment showed that the 1-bp deletion created a frameshift mutation within exon 10. Insertion of the deleted nucleotide by oligonucleotide site-directed mutagenesis restored function to the corrected mutant gene when transfected into gus(mps)/gus(mps) fibroblasts. We concluded that the frameshift mutation, which introduces a premature stop codon at codon 497 in exon 10, accounts for the molecular, biochemical, and pathological abnormalities associated with the gus(mps) phenotype.

Developmental regulation of a gene that encodes a cysteine-rich intestinal protein and maps near the murine immunoglobulin heavy chain locus.

Mouse and rat small intestinal cDNA libraries were screened for recombinants derived from mRNAs whose concentration changed during the transition from suckling to weaning. cDNAs transcribed from a 570-nucleotide-long mRNA were isolated. Dot blot hybridization analyses of RNA recovered at various stages of rat gastrointestinal ontogeny indicated that the concentration of this mRNA begins to increase during the mid-suckling period, reaching a peak during weaning. There is considerable variation in the relative amount of this mRNA in adult tissues, with highest levels encountered in the rat small intestine and colon. Its concentration in duodenum, jejunum, and ileum is approximately the same. It is more concentrated in villi than in crypts. The rat mRNA encodes a 77 amino acid, 8.55-kDa polypeptide that has seven cysteine residues. This cysteine-rich intestinal protein (named CRIP) has two internal repeated sequence blocks. Computer-assisted comparisons of CRIP to proteins of known function disclosed that it is homologous to certain ferredoxins. Southern blot analyses revealed that sequences homologous to the rat gene are present in sea squirt, fish, bird, and human DNA, indicating that this gene is highly conserved and that related proteins may be present in many if not all vertebrates. Recombinant inbred mouse strains were utilized to show that the CRIP gene is closely linked to the immunoglobulin heavy chain constant region locus, Igh-c, on chromosome 12. CRIP mRNA is a molecular marker for the suckling-to-weaning transition of rodent intestinal development. The cloned cDNA may be a useful probe for identifying factors that regulate intestinal development during this period.

Mouse sn-glycerol-3-phosphate dehydrogenase: molecular cloning and genetic mapping of a cDNA sequence.

The isozymes of glycerol-3-phosphate dehydrogenase (GPDH; sn-glycerol-3-phosphate:NAD+2-oxidoreductase, EC 1.1.1.8) in tissues of the mouse are coded for by two structural genes, Gdc-1 and Gdc-2, located on chromosomes 15 and 9, respectively. In order to investigate the expression of these genes, we isolated a GPDH cDNA clone from a mRNA preparation isolated from brown adipose tissue. The GPDH cDNA clone was identified by colony hybridization and hybrid selection of a mRNA that was translated in vitro to produce immunoprecipitable GPDH protein. In blot analysis, the GPDH cDNA hybridized to a single mRNA species that migrated at the position of 23S ribosomal RNA. This GPDH cDNA clone was mapped to the Gdc-1 locus by identification of a restriction enzyme polymorphism present in genomic DNA isolated from Gdc-1 congeneic lines of mice.

Fingerprinting genomes by use of PCR with primers that encode protein motifs or contain sequences that regulate gene expression.

PCR primers of arbitrary nucleotide sequence have identified DNA polymorphisms useful for genetic mapping in a large variety of organisms. Although technically very powerful, the use of arbitrary primers for genome mapping has the disadvantage of characterizing DNA sequences of unknown function. Thus, there is no reason to anticipate that DNA fragments amplified by use of arbitrary primers will be enriched for either transcribed or promoter sequences that may be conserved in evolution. For these reasons, we modified the arbitrarily primed PCR method by using oligonucleotide primers derived from conserved promoter elements and protein motifs. Twenty-nine of these primers were tested individually and in pairwise combinations for their ability to amplify genomic DNA from a variety of species including various inbred strains of laboratory mice and Mus spretus. Using recombinant inbred strains of mice, we determined the chromosomal location of 27 polymorphic fragments in the mouse genome. The results demonstrated that motif sequence-tagged PCR products are reliable markers for mapping the mouse genome and that motif primers can also be used for genomic fingerprinting of many divergent species.

A nuclear extract of Xenopus laevis oocytes that accurately transcribes 5S RNA genes.

Xenopus 5S RNA genes in recombinant form with the plasmid pMB9 are transcribed accurately when added to a supernatant fraction obtained from disrupted nuclei of Xenopus laevis oocytes. After an initial 30 min lag period, the rate of synthesis of 5S RNA is constant for at least an hour and synthesis is still detected after 18 hr. As much as 40% of the total RNA synthesized from the recombinant DNA used in these experiments can be 5S RNA. The coding strand of the 5S RNA genes is transcribed at a rate 10 to 15 times greater than the noncoding strand. Plasmid and spacer DNA, however, are also transcribed. What fraction of total RNA synthesized is 5S RNA is strongly affected by DNA concentration, ionic strength and MgCl2 concentration. Inhibition of transcription by intermediate concentrations of alpha-amanitin demonstrates that RNA polymerase III transcribes at least 90% of all RNA synthesized. Adenovirus 2 DNA is also transcribed in the nuclear supernatant by RNA polymerase III. Approximately 15% of the total RNA synthesized migrates in an acrylamide gel as a band of 5.5S RNA and has been identified as virus-associated RNA1 by its oligonucleotide fingerprint.

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.

Characterization of simian virus 40 tsA58 transcriptional intermediates at restrictive temperatures: relationship between DNA replication and transcription.

When nuclei from simian virus 40 (SV40)-infected cells are lysed with Sarkosyl and the chromatin is pelleted, the supernatant fluid contains a nucleoprotein complex capable of synthesizing viral RNA (Laub and Aloni, Virology 75:346-354, 1976; Gariglio and Mousset, FEBS Lett. 56:149-155, 1975). The level of activity of the RNA polymerase in the complex increased during infection in parallel with the amount of viral DNA that had been synthesized. If cells infected at 33 degrees C with the SV40 mutant tsA 58 were shifted to the nonpermissive temperature of 40 degrees C at any time between 18 and 48 h postinfection, no viral DNA replication was detected after 45 min and no new rounds of synthesis were initiated after 20 to 30 min. However, after this shift, polymerase activity associated with the nucleo-protein complex did continue to increase for 5 h, at which time it reached a plateau. There was an increase of RNA synthesized from both the early (E) and late (L) SV40 DNA strands, and there was a threefold increase in the ratio of early-to-late RNA species after the shift. In comparable experiments with cells infected with wild-type virions, no increase in polymerase activity occurred because of the temperature change alone. At 33 degrees C, the relative amount of RNA transcribed from the wild-type E-strand was less than tsA 58 at 33 degrees C and did not increase after a shift to 40 degrees C. The tsA 58 transcriptional complexes extracted from cells grown at 33 degrees C sedimented heterogeneously in sucrose gradients, with a peak near 26S. There were no detectable alterations in the sedimentation properties of the complexes when tsA 58-infected cells were shifted to 40 degrees C for 2 h. We conclude that continued synthesis of viral DNA is not an obligatory prerequisite for maintenance of late viral transcription nor is the sedimentation of the transcriptional complex at 26S related to actively replicating DNA molecules serving as templates for transcription. Further, an increase in late transcription can occur under conditions where reinitiation of viral DNA synthesis is prevented. The increase in the synthesis of early and late RNA at the restrictive temperature without concurrent DNA synthesis is discussed in relationship to the function of the A gene product.