Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells.

Multipotent and self-renewing neural stem cells have been isolated in culture, but equivalent cells have not yet been prospectively identified in neural tissue. Using cell surface markers and flow cytometry, we have isolated neural crest stem cells (NCSCs) from mammalian fetal peripheral nerve. These cells are phenotypically and functionally indistinguishable from NCSCs previously isolated by culturing embryonic neural tube explants. Moreover, in vivo BrdU labeling indicates that these stem cells self-renew in vivo. NCSCs freshly isolated from nerve tissue can be directly transplanted in vivo, where they generate both neurons and glia. These data indicate that neural stem cells persist in peripheral nerve into late gestation by undergoing self-renewal. Such persistence may explain the origins of some PNS tumors in humans.

Isolation and characterization of mammalian homologs of the Drosophila gene glial cells missing.

The glial cells missing (gcm) gene in Drosophila encodes a transcription factor that determines the choice between glial and neuronal fates. We report here the isolation of two mammalian gcm homologs, Gcm1 and Gcm2, and the characterization of their expression patterns during embryonic development. Although Gcm2 is expressed in neural tissues at a low level, the major sites of expression for both of the mammalian genes are nonneural, suggesting that the functions of the mammalian homologs have diverged and diversified. However, when expressed ectopically, Gcm1 can substitute functionally for Drosophila gcm by transforming presumptive neurons into glia. Thus, certain biochemical properties, although not the specificity of the tissue in which the gene is expressed, have been conserved through the evolution of the Gcm gene family.

Nucleotide sequence of human adenovirus type 12 DNA: comparative functional analysis.

A fresh inoculum of human adenovirus type 12 (Ad12) was obtained from the American Type Culture Collection and passaged once on human embryonic kidney cells, and Ad12 DNA was prepared from the first-passage yield to avoid higher passages which might have generated host-virus DNA recombinants. The 18 PstI fragments of Ad12 DNA were cloned into the pBluescript KS vector, and the entire nucleotide sequence of both strands from all 18 fragments was determined by using successive oligodeoxyribonucleotide primers. Ad12 DNA extends over 34,125 nucleotide pairs, and its molecular weight is calculated to be about 22 x 10(6). The nucleotide sequence of Ad12 DNA was subjected to computer analyses that determined possible open reading of frames on the two strands, the leader sequences, the position of the virus-associated RNA coding region, possible TATA, and polyadenylation signals. The distribution of the Ad12 open reading frames was similar to that in the previously sequenced Ad2 DNA, but there were also distinct differences. Ad12 DNA has an inverted terminal redundancy of 161 nucleotides, compared with 102 nucleotides in Ad2 DNA. There were stretches of sequence identity between Ad2 and Ad12 DNAs at both termini; the overall sequence similarity between the two viral genomes ranged between 59% (polypeptide IX) and 77% (in the E2 region), with high homology also in the sequences for the adenovirus DNA polymerase.

A unique mitigator sequence determines the species specificity of the major late promoter in adenovirus type 12 DNA.

Human adenovirus type 12 (Ad12) cannot replicate in hamster cells, whereas human cells are permissive for Ad12. Ad12 DNA replication and late-gene and virus-associated RNA expression are blocked in hamster cells. Early Ad12 genes are transcribed, and the viral DNA can be integrated into the host genome. Ad12 DNA replication and late-gene transcription can be complemented in hamster cells by E1 functions of Ad2 or Ad5, for which hamster cells are fully permissive (for a review, see W. Doerfler, Adv. Virus Res. 39:89-128, 1991). We have previously demonstrated that a 33-nucleotide mitigator sequence, which is located in the downstream region of the major late promoter (MLP) of Ad12 DNA, is responsible for the inactivity of the Ad12 MLP in hamster cells (C. Zock and W. Doerfler, EMBO J. 9:1615-1623, 1990). A similar negative regulator has not been found in the MLP of Ad2 DNA. We have now studied the mechanism of action of this mitigator element. The results of nuclear run-on experiments document the absence of MLP transcripts in the nuclei of Ad12-infected BHK21 hamster cells. Surprisingly, the mitigator element cannot elicit its function in in vitro transcription experiments with nuclear extracts from both hamster BHK21 and human HeLa cells. Intact nuclear topology and/or tightly bound nuclear elements that cannot be eluted in nuclear extracts are somehow required for recognition of the Ad12 mitigator. Electrophoretic mobility shift assays have not revealed significant differences in the binding of proteins from human HeLa or hamster BHK21 cells to the mitigator sequence in the MLP of Ad12 DNA or to the corresponding sequence in Ad2 DNA. We have converted the sequence of the mitigator in the MLP of Ad12 DNA to the equivalent sequence in the MLP of Ad2 DNA by site-directed mutagenesis. This construct was not active in hamster cells. When the Ad12 mitigator, on the other hand, was inserted into the Ad2 MLP, the latter's function in hamster cells was not compromised. Deletions in the 5' upstream region of the Ad12 MLP have provided evidence for the existence of additional sequences that codetermine the deficiency of the Ad12 MLP in hamster cells. The amphifunctional YY1 protein from HeLa cells can bind specifically to the mitigator and to upstream elements of the MLP of Ad12 DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

A mitigator sequence in the downstream region of the major late promoter of adenovirus type 12 DNA.

Human adenovirus type 12 (Ad12) replicates in permissive human host cells, but undergoes an abortive infection cycle in non-permissive hamster cells. Ad12 DNA cannot replicate and late viral genes are not expressed in hamster cells, whereas most of the early viral mRNAs are synthesized. We have shown previously that the major late promoter of Ad12 DNA (Ad12 MLP; nucleotides -228 to +435 relative to nucleotide +1 as the site of transcriptional initiation) does not function in uninfected or in Ad12-infected hamster BHK21 cells. The transcriptional defect of Ad12 DNA in hamster cells has thus been, at least partly, localized to the viral MLP. As expected, this construct is active in permissive human cells. Here, we show that the sequence between nucleotides +249 and +435 in the Ad12 MLP is in some way responsible for the late transcriptional block of this promoter in hamster cells. An Ad12 MLP--CAT construct comprising nucleotides -228 to +248 shows striking activity in hamster cells, and its activity is very markedly enhanced in Ad2- or Ad12-infected hamster or human cells compared with the nucleotide -228 to +435 construct. By using exonuclease Bal31, a series of Ad12 MLP--CAT gene assemblies were constructed which carry deletions of increasing lengths in the downstream part of the Ad12 MLP. Activity measurements of these constructs in BHK21 and in HeLa cells have located the presumptive mitigator element to the Ad12 sequence between nucleotides +320 and +352 of the MLP. It is also demonstrated that in the nucleotide -228 to +248 MLP construct, transcription is initiated at the authentic Ad12 MLP cap site after the transfection of both hamster and human cells. The localization of this cap site in the nucleotide sequence of the Ad12 MLP indicates the similarity to the comparable start site in the MLP of Ad2 DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Integration of foreign DNA into mammalian genome can be associated with hypomethylation at site of insertion.

The methylation patterns in the genome of mammalian cells are remarkably stable, although occasional changes are observed. In mammalian cells, the non-methylated DNA of human adenovirions (Günthert et al., 1976) undergoes de novo methylation after integration into the host hamster genome (Sutter et al., 1978). The establishment of these specific patterns of methylation in the integrated adenovirus sequences (Sutter and Doerfler, 1979, 1980) requires a considerable number of cell divisions after integration (Kuhlmann and Doerfler, 1982, 1983). Recently, we have reported the analysis of the site of linkage between the left terminus of adenovirus type 12 (Ad12) DNA and unique hamster DNA in the Ad12-induced tumor T1111(2) (Lichtenberg et al., 1987). In what way, if any, are the methylation patterns of the adjacent cellular DNA affected by the insertion of unmethylated foreign (adenoviral) DNA? In normal hamster kidney and spleen DNA and in several Ad12-transformed hamster cell lines, this preinsertion sequence is completely methylated at the 5'-CCGG-3' (HpaII) and 5'-GCGC-3' (HhaI) sequences. The same preinsertion sequences in the DNA of cell line BHK21 and on the non-occupied chromosome in the tumor cell line H1111(2) in passage 9 (p9) are almost completely methylated. In contrast, the same sequence on the chromosome, that carries the integrated Ad12 DNA sequence in the tumor T1111(2), is unmethylated at the 5'-CCGG-3' and 5'-GCGC-3' sequences, as are the abutting Ad12 DNA sequences. Thus, the insertion of unmethylated foreign DNA can lead to the hypomethylation of the flanking cellular DNA in the target sequences.

Reactivation of the methylation-inactivated late E2A promoter of adenovirus type 2 by E1A (13 S) functions.

The inactivating effect of sequence-specific promoter methylations was extensively studied by using the late E2A promoter of adenovirus type 2 (Ad2) DNA. The modification of the three 5' CCGG 3' sequences at nucleotides +24, +6 and -215, relative to the cap site in this promoter, sufficed to silence the gene in transient expression either in Xenopus laevis oocytes or in mammalian cells, and after the fixation of the E2A promoter-chloramphenicol-acetyltransferase (CAT) gene construct in the genome of hamster cells. It will now be demonstrated that the inactivation of the late promoter of Ad2 DNA can be reversed by transactivating functions that are encoded in the 13S messenger RNA of the E1A region of Ad2 DNA. The reactivation of a methylation-inactivated eukaryotic promoter by transactivating functions has general significance in that the value of a regulatory signal can be fully realized only by its controlled reversibility. It was demonstrated in transient expression experiments that the 5' CCGG 3'-methylated late E2A promoter was at least partly reactivated in cell lines constitutively expressing the E1 region of Ad2 or of adenovirus type 5 (Ad5) DNA. The reactivation led to transcriptional initiation at the authentic cap sites of the late E2A promoter and was not associated with promoter demethylation, at least not in both DNA complements. Reactivation of the methylation-inactivated E2A promoter could also be demonstrated in two BHK21 cell lines (mc14 and mc20), which carried the late E2A promoter-CAT gene assembly in an integrated form. In these cell lines the late E2A promoter was methylated and the CAT gene was not expressed. By transfection of cell lines mc14 and mc20, the reactivating functions were shown to reside in the pAd2E1A-13 S cDNA clone of Ad2 DNA. The pAd2E1A-12 S cDNA clone or the pAd2E1B clone showed no reactivating function. These findings implicated the E1A 289 amino acid residue protein of Ad2, a well-known transactivator, as the reactivating function of the endogenous, previously dormant, late E2A promoter-CAT gene assembly. The methylated promoter was not demethylated, at least not in both complements, and it was shown that reactivation of the methylated promoter entailed transcriptional initiation at the authentic late E2A cap site. Since E1A and E1B jointly had a more pronounced effect, it was conceivable that genes in both regions acted together in the abrogation of the inhibitory effect of promoter methylations in the late E2A promoter.

Insertion of adenovirus type 12 DNA in the vicinity of an intracisternal A particle genome in Syrian hamster tumor cells.

In the adenovirus type 12 (Ad12)-induced hamster tumor T1111(2) about 10 Ad12 genome equivalents were integrated at different sites. One of the integrated copies proved unstable and was lost from the cellular genome or rearranged upon passage of the cell line, H1111(2), established from this tumor. This unstable site of junction between the left terminus of Ad12 DNA and hamster DNA and the preinsertion site from BHK21 hamster cells was cloned, sequenced, and analyzed. The junction site showed several peculiarities. At the left terminus of Ad12 DNA, the first 64 nucleotides were deleted. At a distance of 127 nucleotides to the left from this junction site, an internal dispersed fragment of Ad12 DNA comprising nucleotides 1290 to 1361 of the authentic Ad12 DNA sequence was inserted into cellular DNA in an inverted orientation relative to the complete Ad12 genome that was located in its vicinity. The 127-nucleotide sequence between the intact Ad12 genome and the separate 72-base-pair (bp) Ad12 DNA fragment was cellular, but it was not identical to the preinsertion sequence at this location. The sequences flanking the termini of the dispersed 72-bp Ad12 DNA fragment were characterized by direct repeats of 9 or 10 nucleotides. To the left of Ad12 nucleotide 1361 in the separate 72-bp fragment, about 620 cellular nucleotides followed which were identical at the occupied and at the preinsertion sites. It was conceivable that the separate 72-bp Ad12 DNA fragment and the cellular sequence of 127 bp to its right had been transposed en bloc from another unknown location. Abutting the 620 nucleotides of cellular DNA to the left of this block, the 3'-terminal sequence of an endogenous, intracisternal A particle (IAP) genome of hamster cells was detected. The possible significance of the proximity of an IAP sequence to an inserted Ad12 genome with respect to the transformation event, to the instability at this site, or to the transcriptional activity of this region is not known. The 620 bp of cellular DNA between the 72-bp Ad12 DNA fragment and the end of the long terminal repeat of the hamster IAP sequence was apparently of a unique type. Transcriptional activity was not found in the approximate region between nucleotides -620 (to the left) and +350 (to the right) relative to the site of Ad12 DNA insertion, but was found outside these boundaries.