scholarly journals S-phase-specific transcription regulatory elements are present in a replication-independent testis-specific H2B histone gene.

1989 ◽  
Vol 9 (3) ◽  
pp. 1005-1013 ◽  
Author(s):  
I Hwang ◽  
C B Chae
Keyword(s):  
Dna Synthesis ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
S Phase ◽  
Upstream Region ◽  
Base Pairs ◽  
Regulated Expression ◽  
Dna Fragment ◽  
Pachytene Spermatocytes

The testis-specific H2B histone (TH2B) gene is expressed in pachytene spermatocytes during meiotic prophase I in the absence of any significant DNA synthesis. Unlike somatic histones, synthesis of testis-specific histones is not affected by inhibitors of DNA synthesis. A genomic rat TH2B gene was cloned by using a DNA fragment derived from TH2B cDNA as a probe. Expression of the cloned TH2B was investigated by gene transfer experiments. From these studies, we found that the 5' upstream region of the cloned TH2B gene contained S-phase-specific transcription elements which regulated expression of a reporter gene in an S-phase-specific manner. The S-phase-regulatory element was found to be located in two regions containing CCAAT elements between -153 and -110 base pairs (bp) and an octamer element (ATTTGCAT) between -109 and -84 bp. The two regions were required for a maximal stimulation of transcription of the cloned TH2B gene in S phase. On the other hand, only the octamer element was reported be important for the S-phase-specific transcription of human H2B gene. Since the synthesis of the TH2B histone is independent of DNA synthesis and specific for pachytene spermatocytes in vivo, the presence of the S-phase-specific transcription regulatory elements in the TH2B gene is surprising.

scholarly journals S-phase-specific transcription regulatory elements are present in a replication-independent testis-specific H2B histone gene

1989 ◽  
Vol 9 (3) ◽  
pp. 1005-1013
Author(s):  
I Hwang ◽  
C B Chae
Keyword(s):  
Dna Synthesis ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
S Phase ◽  
Upstream Region ◽  
Base Pairs ◽  
Regulated Expression ◽  
Dna Fragment ◽  
Pachytene Spermatocytes

The testis-specific H2B histone (TH2B) gene is expressed in pachytene spermatocytes during meiotic prophase I in the absence of any significant DNA synthesis. Unlike somatic histones, synthesis of testis-specific histones is not affected by inhibitors of DNA synthesis. A genomic rat TH2B gene was cloned by using a DNA fragment derived from TH2B cDNA as a probe. Expression of the cloned TH2B was investigated by gene transfer experiments. From these studies, we found that the 5' upstream region of the cloned TH2B gene contained S-phase-specific transcription elements which regulated expression of a reporter gene in an S-phase-specific manner. The S-phase-regulatory element was found to be located in two regions containing CCAAT elements between -153 and -110 base pairs (bp) and an octamer element (ATTTGCAT) between -109 and -84 bp. The two regions were required for a maximal stimulation of transcription of the cloned TH2B gene in S phase. On the other hand, only the octamer element was reported be important for the S-phase-specific transcription of human H2B gene. Since the synthesis of the TH2B histone is independent of DNA synthesis and specific for pachytene spermatocytes in vivo, the presence of the S-phase-specific transcription regulatory elements in the TH2B gene is surprising.

Cloning of nascent monkey DNA synthesized early in the cell cycle

1985 ◽  
Vol 5 (4) ◽  
pp. 721-727
Author(s):  
G Kaufmann ◽  
M Zannis-Hadjopoulos ◽  
R G Martin
Keyword(s):  
Dna Synthesis ◽  
Animal Cell ◽  
S Phase ◽  
Cell Replication ◽  
Mung Bean Nuclease ◽  
Base Pairs ◽  
African Green Monkey Kidney ◽  
Green Monkey ◽  
Cellular Dna

To study the structure and complexity of animal cell replication origins, we have isolated and cloned nascent DNA from the onset of S phase as follows: African green monkey kidney cells arrested in G1 phase were serum stimulated in the presence of the DNA replication inhibitor aphidicolin. After 18 h, the drug was removed, and DNA synthesis was allowed to proceed in vivo for 1 min. Nuclei were then prepared, and DNA synthesis was briefly continued in the presence of Hg-dCTP. The mercury-labeled nascent DNA was purified in double-stranded form by extrusion (M. Zannis-Hadjopoulos, M. Perisco, and R. G. Martin, Cell 27:155-163, 1981) followed by sulfhydryl-agarose affinity chromatography. Purified nascent DNA (ca. 500 to 2,000 base pairs) was treated with mung bean nuclease to remove single-stranded ends and inserted into the NruI site of plasmid pBR322. The cloned fragments were examined for their time of replication by hybridization to cellular DNA fractions synthesized at various intervals of the S phase. Among five clones examined, four hybridized preferentially with early replicating fractions.

scholarly journals Cloning of nascent monkey DNA synthesized early in the cell cycle.

1985 ◽  
Vol 5 (4) ◽  
pp. 721-727 ◽  
Author(s):  
G Kaufmann ◽  
M Zannis-Hadjopoulos ◽  
R G Martin
Keyword(s):  
Dna Synthesis ◽  
Animal Cell ◽  
S Phase ◽  
Cell Replication ◽  
Mung Bean Nuclease ◽  
Base Pairs ◽  
African Green Monkey Kidney ◽  
Green Monkey ◽  
Cellular Dna

To study the structure and complexity of animal cell replication origins, we have isolated and cloned nascent DNA from the onset of S phase as follows: African green monkey kidney cells arrested in G1 phase were serum stimulated in the presence of the DNA replication inhibitor aphidicolin. After 18 h, the drug was removed, and DNA synthesis was allowed to proceed in vivo for 1 min. Nuclei were then prepared, and DNA synthesis was briefly continued in the presence of Hg-dCTP. The mercury-labeled nascent DNA was purified in double-stranded form by extrusion (M. Zannis-Hadjopoulos, M. Perisco, and R. G. Martin, Cell 27:155-163, 1981) followed by sulfhydryl-agarose affinity chromatography. Purified nascent DNA (ca. 500 to 2,000 base pairs) was treated with mung bean nuclease to remove single-stranded ends and inserted into the NruI site of plasmid pBR322. The cloned fragments were examined for their time of replication by hybridization to cellular DNA fractions synthesized at various intervals of the S phase. Among five clones examined, four hybridized preferentially with early replicating fractions.

Regulatory elements mediating transcription from the Drosophila melanogaster actin 5C proximal promoter

1990 ◽  
Vol 10 (1) ◽  
pp. 206-216
Author(s):  
Y T Chung ◽  
E B Keller
Keyword(s):  
Drosophila Melanogaster ◽  
Transient Expression ◽  
Promoter Activity ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Proximal Promoter ◽  
Base Pairs ◽  
Chloramphenicol Acetyltransferase Gene ◽  
Constitutive Synthesis

The major cytoskeletal actin gene of Drosophila melanogaster, the actin 5C gene, has two promoters, the proximal one of which controls constitutive synthesis of actin in all growing tissues. To locate regulatory elements required for constitutive activity of the proximal promoter, mutants of this promoter were fused to the bacterial chloramphenicol acetyltransferase gene and assayed for transient expression activity in cultured Drosophila embryonic Schneider line 2 cells. An essential regulatory element has been located 313 base pairs upstream from the cap site. Deletion of this element lowered expression to one-third of the wild-type level. The element has the sequence AAGTTGTAGTTG, as shown by protein-binding footprinting with the reagent methidiumpropyl-EDTA-Fe(II). This element is probably not a general one, since it was not detected in a search of the published 5'-flanking sequences of 27 Drosophila genes. In addition to this regulatory element, there are five GAGA elements in the actin 5C proximal promoter, some or all of which are essential for the promoter activity as shown by an in vivo competition assay. Although this promoter has no classical TATA element, there is an essential promoter region about 35 base pairs upstream from the cap site that could be a TATA surrogate. The promoter also shows sequences homologous to the alcohol dehydrogenase factor 1-binding site and to the core of the vertebrate serum response element, but mutations of these sites did not affect promoter activity in transient expression assays.

scholarly journals Characterization of the S-phase-specific transcription regulatory elements in a DNA replication-independent testis-specific H2B (TH2B) histone gene.

1990 ◽  
Vol 10 (2) ◽  
pp. 585-592 ◽  
Author(s):  
I W Hwang ◽  
K Lim ◽  
C B Chae
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Biological Significance ◽  
Regulatory Elements ◽  
S Phase ◽  
Phase Cell ◽  
Dependent Manner ◽  
Base Pairs ◽  
Pachytene Spermatocytes ◽  
Cloned Gene

The testis-specific H2B histone (TH2B) gene is expressed in pachytene spermatocytes of meiotic prophase I during rat spermatogenesis. The TH2B RNA and histones are not synthesized in any other tissues, and the synthesis is independent of DNA replication. However, the cloned TH2B gene has two DNA sequence elements which stimulate transcription of the cloned gene in an S-phase-dependent manner when introduced into somatic cells. The factors interacting with the two elements, CCAAT at -127 base pairs and octamer ATTTGCAT at -93 base pairs, interact with each other to bring about a maximum stimulation of S-phase-dependent transcription. The level of CCAAT and octamer-binding proteins is unchanged during the cell cycle, and the S-phase-dependent transcription of TH2B and endogenous mouse H2B genes does not require synthesis of new proteins during the S phase. Cell cycle-specific posttranslational modification of regulatory proteins may be responsible for the S-phase-dependent transcription of H2B histone genes. The biological significance of the presence of S-phase-specific transcription regulatory elements in the DNA replication-independent and tissue-specific TH2B gene is not known.

scholarly journals Characterization of the S-phase-specific transcription regulatory elements in a DNA replication-independent testis-specific H2B (TH2B) histone gene

1990 ◽  
Vol 10 (2) ◽  
pp. 585-592
Author(s):  
I W Hwang ◽  
K Lim ◽  
C B Chae
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Biological Significance ◽  
Regulatory Elements ◽  
S Phase ◽  
Phase Cell ◽  
Dependent Manner ◽  
Base Pairs ◽  
Pachytene Spermatocytes ◽  
Cloned Gene

The testis-specific H2B histone (TH2B) gene is expressed in pachytene spermatocytes of meiotic prophase I during rat spermatogenesis. The TH2B RNA and histones are not synthesized in any other tissues, and the synthesis is independent of DNA replication. However, the cloned TH2B gene has two DNA sequence elements which stimulate transcription of the cloned gene in an S-phase-dependent manner when introduced into somatic cells. The factors interacting with the two elements, CCAAT at -127 base pairs and octamer ATTTGCAT at -93 base pairs, interact with each other to bring about a maximum stimulation of S-phase-dependent transcription. The level of CCAAT and octamer-binding proteins is unchanged during the cell cycle, and the S-phase-dependent transcription of TH2B and endogenous mouse H2B genes does not require synthesis of new proteins during the S phase. Cell cycle-specific posttranslational modification of regulatory proteins may be responsible for the S-phase-dependent transcription of H2B histone genes. The biological significance of the presence of S-phase-specific transcription regulatory elements in the DNA replication-independent and tissue-specific TH2B gene is not known.

scholarly journals Regulatory elements mediating transcription from the Drosophila melanogaster actin 5C proximal promoter.

1990 ◽  
Vol 10 (1) ◽  
pp. 206-216 ◽  
Author(s):  
Y T Chung ◽  
E B Keller
Keyword(s):  
Drosophila Melanogaster ◽  
Transient Expression ◽  
Promoter Activity ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Proximal Promoter ◽  
Base Pairs ◽  
Chloramphenicol Acetyltransferase Gene ◽  
Constitutive Synthesis

The major cytoskeletal actin gene of Drosophila melanogaster, the actin 5C gene, has two promoters, the proximal one of which controls constitutive synthesis of actin in all growing tissues. To locate regulatory elements required for constitutive activity of the proximal promoter, mutants of this promoter were fused to the bacterial chloramphenicol acetyltransferase gene and assayed for transient expression activity in cultured Drosophila embryonic Schneider line 2 cells. An essential regulatory element has been located 313 base pairs upstream from the cap site. Deletion of this element lowered expression to one-third of the wild-type level. The element has the sequence AAGTTGTAGTTG, as shown by protein-binding footprinting with the reagent methidiumpropyl-EDTA-Fe(II). This element is probably not a general one, since it was not detected in a search of the published 5'-flanking sequences of 27 Drosophila genes. In addition to this regulatory element, there are five GAGA elements in the actin 5C proximal promoter, some or all of which are essential for the promoter activity as shown by an in vivo competition assay. Although this promoter has no classical TATA element, there is an essential promoter region about 35 base pairs upstream from the cap site that could be a TATA surrogate. The promoter also shows sequences homologous to the alcohol dehydrogenase factor 1-binding site and to the core of the vertebrate serum response element, but mutations of these sites did not affect promoter activity in transient expression assays.

scholarly journals A MYBL2 complex for RRM2 transactivation and the synthetic effect of MYBL2 knockdown with WEE1 inhibition against colorectal cancer

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Qian Liu ◽  
Lijuan Guo ◽  
Hongyan Qi ◽  
Meng Lou ◽  
Rui Wang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Dna Synthesis ◽  
Ectopic Expression ◽  
Building Blocks ◽  
Small Subunit ◽  
S Phase ◽  
Clinical Samples ◽  
Dependent Manner

AbstractRibonucleotide reductase (RR) is a unique enzyme for the reduction of NDPs to dNDPs, the building blocks for DNA synthesis and thus essential for cell proliferation. Pan-cancer profiling studies showed that RRM2, the small subunit M2 of RR, is abnormally overexpressed in multiple types of cancers; however, the underlying regulatory mechanisms in cancers are still unclear. In this study, through searching in cancer-omics databases and immunohistochemistry validation with clinical samples, we showed that the expression of MYBL2, a key oncogenic transcriptional factor, was significantly upregulated correlatively with RRM2 in colorectal cancer (CRC). Ectopic expression and knockdown experiments indicated that MYBL2 was essential for CRC cell proliferation, DNA synthesis, and cell cycle progression in an RRM2-dependent manner. Mechanistically, MYBL2 directly bound to the promoter of RRM2 gene and promoted its transcription during S-phase together with TAF15 and MuvB components. Notably, knockdown of MYBL2 sensitized CRC cells to treatment with MK-1775, a clinical trial drug for inhibition of WEE1, which is involved in a degradation pathway of RRM2. Finally, mouse xenograft experiments showed that the combined suppression of MYBL2 and WEE1 synergistically inhibited CRC growth with a low systemic toxicity in vivo. Therefore, we propose a new regulatory mechanism for RRM2 transcription for CRC proliferation, in which MYBL2 functions by constituting a dynamic S-phase transcription complex following the G1/early S-phase E2Fs complex. Doubly targeting the transcription and degradation machines of RRM2 could produce a synthetic inhibitory effect on RRM2 level with a novel potential for CRC treatment.

SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase

1993 ◽  
Vol 13 (9) ◽  
pp. 5829-5842
Author(s):  
P Zheng ◽  
D S Fay ◽  
J Burton ◽  
H Xiao ◽  
J L Pinkham ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Synthesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Low Frequency ◽  
S Phase ◽  
Upstream Region ◽  
Specific Gene

SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.

Ecdysterone regulatory elements function as both transcriptional activators and repressors

1991 ◽  
Vol 11 (4) ◽  
pp. 1846-1853
Author(s):  
L Dobens ◽  
K Rudolph ◽  
E M Berger
Keyword(s):  
Regulatory Element ◽  
Regulatory Elements ◽  
Upstream Region ◽  
Transcriptional Activators ◽  
Reporter Plasmid ◽  
Bacterial Gene ◽  
Simplex Virus ◽  
Very High ◽  
Cat Gene ◽  
Cat Expression

A synthetic, 23-bp ecdysterone regulatory element (EcRE), derived from the upstream region of the Drosophila melanogaster hsp27 gene, was inserted adjacent to the herpes simplex virus thymidine kinase promoter fused to a bacterial gene for chloramphenicol acetyltransferase (CAT). Hybrid constructs were transfected into Drosophila S3 cells and assayed for ecdysterone-inducible CAT expression. In the absence of ecdysterone a tandem pair of EcREs repressed the high constitutive level of CAT activity found after transfection with the parent reporter plasmid alone. After hormone addition very high levels of CAT activity were observed. Insertion of the EcRE pair 3' of the CAT gene also led to high levels of ecdysterone-induced CAT expression, but the repression of high constitutive levels of CAT activity failed to occur. The EcRE-CAT construct was cotransfected with plasmids containing tandem 10-mers or 40-mers of the EcRE but lacking a reporter gene. These additional EcREs led to a reduced level of ecdysterone-induced CAT activity and to an elevation of basal CAT activity in the absence of hormone. The data suggest that the receptor binds to the EcRE in the absence of hormone, blocking basal transcription from a constitutive promoter. In the presence of ecdysterone, receptor-hormone binding to the EcRE leads to greatly enhanced transcription.

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