I report a characterization of several alleles of the myospheroid (mys) gene which encodes the beta-chain of the Drosophils PS integrins. Genetic analysis revealed that the mysˣᴿ, mutation is antimorphic and the mysˣᴺ mutation is hypomorphic. Protein was detected on Western blots from hemizygous mysˣᴿ and mysˣᴺ animals. No integrin beta-subunit was detected in in situ immunofluorescence assays in mysˣᴿ embryos. However, antigen was detected in a small subset of muscle attachment sites in mysˣᴺ embryos. mysˣᴳ and mysˣᴮ alleles behaved, genetically, as null alleles and no protein was detected on Western blots or in immunofluorescence assays. Complementation tests between mysᵗˢ¹, mysᵗˢ³, and the other lethal mys alleles unusual results which suggest that mys may be a transvecting locus.

We have identified a region in the coding sequence (CRAS) of two replication-dependent histone genes, a H2a.2 and a H3.2, the deletion of which causes a 20-fold drop in expression. In vivo expression data obtained after transfection of in-frame oligonucleotide-directed deletions in the H3.2 CRAS show that there are multiple areas in the CRAS which act to increase expression of the intact gene. DNAase I protection of a sequence designated the CRAS alpha box is present in the CRAS of four classes of histone genes--H2a, H2b, H3 and H4. Deletion of the alpha box causes a 5-fold drop in expression of the H3.2 gene. The comparable "CRAS" region of a H3.3 gene, a nonreplication-dependent variant, was used as a non-specific competitor. Although the coding region is highly conserved among classes of histone genes, the H3.3 "alpha box" sequence contains 5 base changes out of 7. UV crosslinking of the protein bound H3.2 CRAS alpha sequence identifies a protein doublet migrating at 80 and 90 kD on SDS-polyacrylamide gels. Binding to the alpha box is regulated by the phosphorylation state of the nuclear protein that binds the alpha sequence. Dephosphorylation of the CRAS alpha protein is necessary for binding to the target sequence. The role of the CRAS-binding proteins in cell cycle regulation of histone gene expression was studied by preparing G1 and S phase nuclear extracts from synchronous populations of CHO cells. Binding of CRAS alpha protein to duplex oligonucleotides containing the consensus sequence is greatly elevated in S phase compared to G1. / Source: Dissertation Abstracts International, Volume: 55-04, Section: B, page: 1295. / Major Professor: William F. Marzluff. / Thesis (Ph.D.)--The Florida State University, 1994.

Sequences homologous to a putative origin of DNA replication from Xenopus laevis have been cloned and sequenced. The prototype, Xori, has been demonstrated to enhance the replication of vector molecules microinjected into Xenopus eggs. Thus, it was of interest to isolate additional clones and to compare their sequences in order to determine if common sequence motifs exist, which might provide some insight into gene regulation and DNA replication. Including Xori, six clones were compared. Two of the clones lie in the globin gene cluster; one 5' to the adult (alpha)1 gene, and the other in the second intron of the tadpole (alpha)1 gene. In all clones, the homologous region begins at the same site, but the lengths of the common regions vary from 142 bp to over 300 bp due to heterogeneous 3' ends. Some of the repeats are bracketed by direct and/or inverted repeats, and relatively large palindromes were found 5' to the common region in some clones. These characteristics, and the presence of a repeat 5' to one of a pair of duplicated (alpha)1 genes suggests that Xori may be capable of transposition. The homologous regions are 20 to 30 percent divergent, but contain a number of sequences of interest. Five clones contain 17 elements which are highly homologous to the yeast autonomously replicating sequence, as well as a region of 25 bp which is similar to the SV40 enhancer. However, following transfection of cultured cells, plasmids harboring repeats are progressively lost, and apparently do not replicate. Although the repeats are transcribed at gastrulation, they are not transcribed in oocytes, blood, or in kidney cells in culture, nor are they associated with the nuclear matrix. Implications of these data are discussed. / Source: Dissertation Abstracts International, Volume: 47-06, Section: B, page: 2319. / Thesis (Ph.D.)--The Florida State University, 1986.

Genes coding for replication-dependent, partially replication-dependent, and replication-independent histones have been isolated from mouse. These genes were isolated from libraries of mouse DNA in lambda bacteriophage. Three previously-isolated mouse histone genes, several heterologous histone genes, and synthetic oligonucleotides were used to identify the clones. The four replication-dependent and two partially replication-dependent genes have been sequenced. Transcripts of these genes from mouse myeloma cells, from C127 mouse fibroblasts, and from differentiating murine erythroleukemia cells have been characterized using the S1 nuclease assay. Transcripts of several of the replication-independent genes from differentiating murine erythroleukemis cells have been studied, and one of these genes has been sequenced. There are no intervening sequences in the coding region of this gene, but there may be intervening sequences in the 5' flanking region. Primer extension studies have been used to determine the length of the transcript from the genes for replication-independent histones. The replication-dependent and partially replication-dependent histone genes are clustered, and the clusters contain members of only one expression class. Only genes coding for the core histones have been found in these clusters. The replication-independent H3 genes appear to be isolated from other histone genes. / Source: Dissertation Abstracts International, Volume: 47-01, Section: B, page: 0083. / Thesis (Ph.D.)--The Florida State University, 1986.

Control of the timing of replication within S-phase of chromosomes or chromosome bands has long been suspected to be a general mechanism of compartmentalizing the genome for differential gene expression. I have developed a method that provides both a direct probe for active and inactive regions of the chromosome and a means to determine the time of replication of these individual sites. By using a retrovirus shuttle vector which contains a selectable marker gene (HSV-tk), to infect tissue culture cells, it is possible to introduce the same single copy sequence in many different chromosomal locations. I have isolated and characterized a series of cell clones which have single or a small number of either active or cryptic copies of the marker gene. To determine the time of replication of the marker sequence, asynchronous or synchronized cell cultures were pulse labeled with bromo-deoxy-uridine. Cells were fractionated according to their position in S-phase by mitotic collection or separately pulse labeled cultures (for synchronized cells). BrdU containing DNA was then degraded selectively by exposure to Hoechst 33257 dye, long wave UV light and S1 nuclease, which makes it possible to analyze each cell fraction with regard to BrdU substitution in the marker sequence. One can thus infer the time of replication of a specific single copy sequence. All clones analyzed, both expressing and cryptic clones, replicate the SW 272 sequence in early S-phase. The findings suggest, that this retrovirus does not integrate randomly into the cellular genome with respect to replication compartments. In one clone the cryptic copy of the viral Tk-gene, could be activated by treatment with 5-aza-cytidine, implying a role of cytosine methylation in regulation of expression in this case. / Source: Dissertation Abstracts International, Volume: 47-07, Section: B, page: 2773. / Thesis (Ph.D.)--The Florida State University, 1986.

Sequences at both 5$\sp\prime$ and 3$\sp\prime$ ends of mouse histone genes contribute to the expression of the gene. The effect of 5$\sp\prime$ sequences of H2a(614) and H3.2(614) is presumably on the rate of transcription. The 3$\sp\prime$ sequences required for high expression of the mouse H2a(614) gene are the same as the sequences required for 3$\sp\prime$ end formation. When these sequences are substituted for the 3$\sp\prime$ end of the poorly expressed H2a(291) gene, the expression of the H2a(291) gene is increased 5-fold. A 65 nucleotide fragment containing the H2a(614) 3$\sp\prime$ processing signal increases the expression of the H2a(291) gene when it is placed in the proper orientation downstream of the H2a(291) 3$\sp\prime$ end, suggesting that the transcript is sequentially processed. In in vitro processing experiments, the different histone 3$\sp\prime$ ends show different processing efficiencies which correlate with the expression in cells. This hairpin loop structure has multiple uses which include transcription termination, mRNA stability and efficiency of processing by the cell to regulate the steady-state level of histone mRNAs. / Source: Dissertation Abstracts International, Volume: 50-05, Section: B, page: 1785. / Major Professor: William F. Marzluff. / Thesis (Ph.D.)--The Florida State University, 1989.

RNA polymerase II transcribes all mRNAs as well as several of the small nuclear RNA (snRNA) genes. Among RNA pol II genes, the U1 snRNA genes and replication dependent histone genes are distinct in their lack of intervening sequences and polyadenylated 3$\sp\prime$ ends. A unique feature of the U1 gene is that U1 3$\sp\prime$end formation requires an snRNA promoter. The promoters used in those experiments were for genes that normally form polyadenylated mRNAs. I constructed chimeric mouse histone-U1 genes to see if the U1 3$\sp\prime$end would be used when coupled to a promoter from a non-polyadenylated transcript. Using an S1 nuclease assay I showed that the transcripts do not form the U1 3$\sp\prime$end, but end at heterogeneous sites, implying that the pol II-histone transcription complex lacks a U1-specific 3$\sp\prime$end-forming activity. / It has also been reported that the transcripts initiating from U1 promoters do not direct synthesis of polyadenylated mRNA. I also constructed chimeric mouse U1-histone genes and showed that U1 promoters with at least 5 base pairs of the coding sequence highly express the histone mRNA. Transcripts of these chimeric genes mapped to the normal U1 start and formed normal histone 3$\sp\prime$ends. / When I combined these genes to make UHU genes (U1 promoter, histone coding, U1 3$\sp\prime$end), 50% of the RNAs had U1 3$\sp\prime$ends, and the other 50% were readthrough transcripts with heterogeneous ends. When I added the histone 3$\sp\prime$end to the UHU gene to make UHUH and UHHU genes, most transcripts had histone ends and were translated efficiently. Experiments comparing the stability and relative amounts of UHU and UHUH RNAs showed that a downstream histone end did not affect the efficiency of U1 3$\sp\prime$end formation, suggesting that U1 3$\sp\prime$ ends are formed by transcription termination, unlike any other pol II genes. When the distance between the U1 promoter and U1 3$\sp\prime$ end was shortened to 146, more U1 ends were formed than histone ends, demonstrating that the coupling of the U1 promoter and U1 3$\sp\prime$ end formation is distance dependent. Long UHUH transcripts with histone ends were transported to the cytoplasm and efficiently translated whereas transcripts of a similar length with U1 ends may have never left the nucleus. This suggests that the histone 3$\sp\prime$ end functions either as a signal for transport from the nucleus to the cytoplasm or as a signal for efficient translation. / Source: Dissertation Abstracts International, Volume: 51-09, Section: B, page: 4195. / Major Professor: William F. Marzluff. / Thesis (Ph.D.)--The Florida State University, 1990.

Satellite 2 is a 300-350 bp tandemly repeated DNA that is highly conserved in different salamander families. While the function of satellite 2 is not known, it has the ability to promote transcription using a self-contained snRNA type of promoter and its transcripts can catalyze their own site-specific cleavage using an extended hammerhead domain. An analysis of chimeric hammerheads indicated that the different stem I extensions were active only in the context of compatible stem II regions. To investigate this compatibility requirement, partial satellite 2 sequences containing the extended hammerhead domain were cloned and characterized from six species representing four Caudate families. Results indicated that satellite 2 elements exist in every Caudate family except Sirenidae. Despite differences in the abundance of this element and its transcripts, the tandemly repeated genomic organization and the tissue specific transcript patterns have been conserved. Comparison of the satellite 2 sequences from different species suggested the existence of at least two groups, and they might evolve at different rates in different families. Sequence analysis also revealed that the species consensus sequence is under selection while individual repeats are free to diverge. However, this selection is not for increasing the cleavage ability of its extended hammerhead. Although each species consensus sequence can form an extended hammerhead that has the same central core and the same secondary structure, these extended hammerheads have very different in vitro cleavage rates. Since the differences are primarily in the stem I and stem II regions, the results suggest that both regions are important for self-cleavage. / Source: Dissertation Abstracts International, Volume: 56-07, Section: B, page: 3611. / Major Professor: Lloyd M. Epstein. / Thesis (Ph.D.)--The Florida State University, 1995.

In the sea urchin there is a tandemly repeated U2 snRNA gene set that is expressed early in embryogenesis and inactivated after blastula stage. In addition, there is a constitutive gene set which is expressed in all cells. A tandemly repeated and a single copy U2 snRNA gene were isolated from the sea urchin L. variegatus. Upon microinjection into sea urchin zygotes, the genes showed temporal expression characteristic of an embryonic and a constitutive gene, respectively. Four cis-acting elements were identified within the promoter of each gene: the TATA box at $-$30, the PSE element at $-$60, an element at $-$100 and an upstream activating sequence. Other than the TATA box there is no sequence homology between the elements from the two genes. The TATA and the PSE element are necessary and sufficient for basal transcription and for selection of the transcription initiation site. Swapping of the embryonic gene PSE element into the constitutive gene promoter converts temporal expression of the constitutive gene into that of the embryonic gene, suggesting that the PSE sequence is responsible for developmental regulation. A protein complex binds to the PSE and TATA elements of the embryonic gene in vitro. In vitro binding correlates with activity of the promoter in vivo. The binding activity is present in blastula nuclei and absent from gastrula nuclei. An 82 kD protein was UV crosslinked to the embryonic gene PSE sequence. The protein has been purified by DNA affinity chromatography. Cloning of its cDNA is a future goal. It is likely that the 82 kD factor is a key factor for developmental regulation of the sea urchin embryonic U2 snRNA genes. / Source: Dissertation Abstracts International, Volume: 52-11, Section: B, page: 5678. / Major Professor: William F. Marzluff, Jr. / Thesis (Ph.D.)--The Florida State University, 1991.

DNA replication-dependent histone genes differ from other genes which are transcribed by RNA polymerase II in that histone genes do not contain introns and have no poly(A) tail at their 3$\sp\prime$ ends. The histone mRNAs end with a conserved stem-loop. This thesis examines the requirements involved in the process of transporting histone mRNA from the nucleus to the cytoplasm. The 3$\sp\prime$ processing signal is required for exporting histone mRNA out of the nucleus. This sequence can be replaced with a polyadenylation signal but not with U1 snRNA 3$\sp\prime$ end. This sequence may also play an important role in promoting the association of histone mRNA with polyribosomes and assist in the initiation of translation of histone mRNA. A 50KD stem-loop binding protein (SLBP) has been identified by RNA mobility shift and UV-crosslinking assays. Different complexes are formed in the nucleus and the cytoplasm with the nuclear complex being larger. The nuclear and cytoplasmic SLBP are the same proteins demonstrated by partial proteolysis. The interaction between the stem-loop RNA and SLBP is extremely sequence dependent. However none of the mutants of stem-loop has a significant effect on transport of the mRNA. Instead the mutations severely reduce the SLBP binding activity and expression of histone mRNA. These suggest that the interaction between the SLBP and stem-loop is critical for histone mRNA processing. An additional nuclear factor has also been identified which is involved in nuclear SLBP-RNA complex and is probably located on the nuclear membrane, suggesting that this factor along with SLBP may function in the process of exporting histone mRNA to the cytoplasm. The dissociation of SLBP from histone mRNA may be the initial step for degradation to take place. The SLBP is recycled after histone mRNA is degraded and return to the nucleus. A model of the functions of SLBP in the metabolism and regulation of histone mRNA is proposed. / Source: Dissertation Abstracts International, Volume: 53-03, Section: B, page: 1209. / Major Professor: William F. Marzluff. / Thesis (Ph.D.)--The Florida State University, 1992.