Regulation of gene function: a comparison of enzyme activity levels in relation to gene dosage in diploids and triploids of Drosophila melanogaster.
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Animals↗
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Results for “gene function”
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The analysis of new short-period circadian rhythm mutants suggests features of D. melanogaster period gene function.
A number of new period gene (per) mutants were generated by in vitro mutagenesis and germ line transformation. Missense mutations were made at amino acid 589, which is altered in the 19 h short-period (per(s)) mutant, and insertion mutations were generated with peptides commonly used for epitope tagging. Most of these new per mutants had short behavioral rhythms. Flies with heteroallelic combinations of these new mutant per genes were found to have "hybrid" periods, i.e., they had values that were usually in between those of the individual alleles. These findings suggest that short-period per mutants are not unusual gain-of-function mutants but rather more traditional loss-of-function mutants that are unable to influence the circadian pacemaker in a proper manner. The data also suggest that the per protein may engage in important intermolecular interactions.
Animals↗
Changes in growth and tumorigenicity following reconstitution of retinoblastoma gene function in various human cancer cell types by microcell transfer of chromosome 13.
Functional loss of the retinoblastoma (RB) gene has been implicated in the initiation or progression of several human tumor types including cancer of the eye, bone, bladder, and prostate. To examine the consequence of adding one RB allele containing its normal regulatory elements back into representative examples of each of these cancer types, as well as to compare the results to those previously reported using various RB complementary DNA constructs, a neomycin resistant marked 13 chromosome was transferred by microcell fusion. Several attempts to obtain RB positive osteosarcoma cells failed. In addition, only one RB positive retinoblastoma clone was isolated. This clone contained many large cells, could not be maintained in long-term culture, and produced only RB negative tumors. Three RB positive bladder cancer cell clones were obtained, all of which grew slower in culture than their RB negative parental counterpart and did not form colonies in soft agar. Tumorigenicity was markedly suppressed in these clones. One clone yielded no tumors, and the other 2 clones produced only one small tumor each, both of which were RB negative. In contrast, the 2 RB positive prostate cancer cell clones isolated had no differences in their cell culture growth properties, including growth in soft agar compared to the parental cells. One of the clones was nontumorigenic, while the other clone produced 4 small tumors, all of which were RB positive. These results indicate that the transfer of one RB allele by microcell transfer produces different levels of growth inhibition as well as tumor suppression, depending on the cell type examined. In the case of prostate cancer, the function of the RB gene in tumor suppression appears to be independent from its growth regulatory function, since no growth inhibition in cell culture was noted in these cells, although tumor suppression was significant.
Alleles↗
Setting limits on homeotic gene function: restraint of Sex combs reduced activity by teashirt and other homeotic genes.
Each of the homeotic genes of the HOM or HOX complexes is expressed in a limited domain along the anterior-posterior axis. Each homeotic protein directs the formation of characteristic structures, such as wings or ribs. In flies, when a heat shock-inducible homeotic gene is used to produce a homeotic protein in all cells of the embryo, only some cells respond by altering their fates. We have identified genes that limit where the homeotic gene Sex combs reduced (Scr) can affect cell fates in the Drosophila embryo. In the abdominal cuticle Scr is prevented from inducing prothoracic structures by the three bithorax complex (BX-C) homeotic genes. However, two of the BX-C homeotic genes, Ultrabithorax (Ubx) and abdominal-A (abd-A), have no effect on the ability of Scr to direct the formation of salivary glands. Instead, salivary gland induction by Scr is limited in the trunk by the homeotic gene teashirt (tsh) and in the last abdominal segment by the third BX-C gene, Abdominal-B (AbdB). Therefore, spatial restrictions on homeotic gene activity differ between tissues and result both from the regulation of homeotic gene transcription and from restraints on where homeotic proteins can function.
Animals↗
Cytoplasmic effect on gene function in Xenopus laevis.
The pigmentation gene of Xenopus laevis is dominant and that of albino aP mutant recessive. Heterologous haploid hybrids are produced by UV inactivation of the egg nuclei during second polar body formation in the mutant sperm-fertilized Xenopus eggs. During development of these hybrids, melanin appeared in the eye and melanophores in the skin at stages comparable to those of the wild type, but much earlier than in the albino mutant. The number and intensity of pigment cells are intermediate between the black Xenopus and albino mutant. While a number of pigment cells remain in the hybrids, those in the albino eventually degenerate. Therefore, the development and maintenance of pigmentation in heterologous hybrids are contributed by Xenopus cytoplasm. Tadpole tail-tips were squashed and stained for chromosome counting. The results show that Xenopus and mutants are diploid (36 chromosomes) and heterologous haploid hybrids have 18 chromosomes.
Animals↗
Adherent cell function in murine T lymphocyte antigen recognition. III. A macrophage-mediated immune response gene function in the mouse.
The I region of the MHC appears to control antigen-specific macrophage-T lymphocyte interaction. The immune response to antigens such as Gl phi 9 are under control of two distinct I subregions, I-A and I-E/I-C. We have asked in a macrophage-dependent, antigen-specific murine T cell proliferation assay whether either or both gene products need be expressed in the antigen-presenting cells. We find that both Ir-Gl phi 9 alpha and beta genes must be expressed and function in the antigen-presenting cell.
Animals↗
Chromosomal protein HMG-14. Identification, characterization, and chromosome localization of a functional gene from the large human multigene family.
The human HMG-14 multigene family is one of the largest retropseudogene families known. To identify and isolate a functional human HMG-14 gene, genomic clones, selected with the cDNA, were screened with a set of 6 oligonucleotides. A single genomic clone was isolated suggesting that the human genome contains few, and perhaps only one, functional genes. An 8882-base pair (bp) genomic clone containing the complete, 6804-bp-long human gene together with 850 bp 5' to the start of transcription and 1228 bp 3' to the end of transcription was sequenced. The gene is comprised of 6 exons ranging in size from 30 to 839 bp, two of which code for the entire DNA binding site of the protein, and has several features typical of "housekeeping" genes. Using human-rodent somatic cell hybrids, the HMG-14 gene was localized to human chromosome 21. A restriction fragment length polymorphism, useful for further analysis and mapping, has been detected. The present article, which describes the first isolation and characterization of a gene coding for chromosomal protein HMG-14, indicates that genes coding for HMG-14 and HMG-17 may share several distinctive characteristics. Comparison with the human and chicken HMG-17 genes reveals that all contain 6 exons, that all have exons of similar size, that all have 5' regions highly enriched in GC residues and that all have features typical of housekeeping genes.
Animals↗
Implementing transgenic and embryonic stem cell technology to study gene expression, cell-cell interactions and gene function.
This review highlights the use of transgenic mice and gene targeting in the study of reproduction, pituitary gene expression, and cell lineage. Since 1980 numerous applications of transgenic animal technology have been reported. Altered phenotypes resulting from transgene expression demonstrated that introduced genes can exert profound effects on animal physiology. Transgenic mice have been important for the study of hormonal and developmental control of gene expression because gene expression in whole animals often requires more DNA sequence information than is necessary for expression in cell cultures. This point is illustrated by studies of pituitary glycoprotein hormone alpha- and beta-subunit gene expression (Kendall et al., Mol Endocrinol 1994; in press [1]. Transgenic mice have also been invaluable for producing animal models of cancer and other diseases and testing the efficacy of gene therapy. In addition, cell-cell interactions and cell lineage relationships have been explored by cell-specific expression of toxin genes in transgenic mice. Recent studies suggest that attenuated and inducible toxins hold promise for future transgene ablation experiments. Since 1987, embryonic stem (ES) cell technology has been used to create numerous mouse strains with targeted gene alterations, contributing enormously to our understanding of the functional importance of individual genes. For example, the unexpected development of gonadal tumors in mice with a targeted disruption of the inhibin gene revealed a potential role for inhibin as a tumor suppressor (Matzuk et al., Nature 1992:360: 313-319 [2]. The transgenic and ES cell technologies will undoubtedly continue to expand our understanding and challenge our paradigms in reproductive biology.
Animals↗
Generation of spCAS9 expressing human mesenchymal stem cell line to study gene function during osteoblast differentiation.
Human bone marrow-derived stromal cells (hMSCs) are a great resource for studying how genes influence cell fate and differentiation into various cell types like osteoblasts, adipocytes, and chondrocytes, among other cell types. However, genetic manipulation of primary hMSCs has been challenging due to their short lifespan and cellular senescence after limited passaging. Their low and unstable transfection efficiency also complicates gene delivery or inactivation, hindering long-term functional studies. The limited lifespan has been effectively solved by immortalizing hMSCs with telomerase reverse transcriptase (hMSCs-TERT). The use of these cells is ideal for functional studies of osteoblast and adipocyte differentiation through genetic manipulation, providing a stable and reliable model. Here, we have engineered a stable CAS9 expressing hMSC-TERT cell line (hMSC-TERTCAS9) via lentiviral transduction. The constitutive expression of spCas9 enables efficient and reproducible gene editing. We demonstrate the potential of these hMSC-TERTCAS9 cells for generating gene disruptions using plasmid delivery of guide RNAs as a fast and efficient strategy for targeted genome editing. The edited cells can be sorted and expanded as single cells to obtain homogenous clonal cell lines with mono- as well as bi-allelic gene deletions, a crucial step for producing reliable experimental results. We further validate this cell line as a powerful tool for studying gene function during hMSC proliferation and differentiation, providing 3 distinct examples of its utility. Through the generation of indels, single-cell sorting, and clonal selection, we have efficiently inactivated the vitamin D receptor and created both larger (256 nucleotides) gene disruptions in Forkhead box protein O1 and precise removals of a small genomic sequence (73 nucleotides) coding for microRNA MIR675. This novel hMSC-TERTCAS9 cell line represents a significant advancement, offering a stable, efficient, and versatile platform for advanced genetic studies, high-throughput screening, and the creation of reliable cellular disease models.
CRISPR-Cas9↗
Study of the higher eukaryotic gene function CDK2 using fission yeast.
In the fission yeast Schizosaccharomyces pombe, cdc2 function is required both in G1 to enter the cell cycle and in G2 to initiate mitosis. In higher eukaryotes, these functions appeared to be shared between several cdc2-like genes including CDK2. Temperature-sensitive mutations in S. pombe cdc2 that arrest the cell cycle in both G1 and G2 phases are not complemented by CDK2. We have used S. pombe to investigate what functions CDK2 can perform. We found that overexpression of the human homologue (HsCDK2) caused cell cycle arrest in G2/M showing that HsCDK2 interfered with mitotic events. Xenopus CDK2 (XlCDK2) overexpression did not cause cell cycle arrest and could rescue the G1 block but not the G2 block of a cdc2-M26 ts strain. A mutant XlCDK2-R33, which is inactive as a kinase, failed to rescue the G1 block, suggesting that the protein kinase activity of CDK2 is required to enter the cell cycle in these circumstances. We designed screens to select mutants that would require XlCDK2 expression for viability, hoping to isolate new gene functions interacting with, or that could be replaced by, XlCDK2 in G1, or new cdc2 mutants altered solely in their G1 role. From these screens several cell cycle mutants were selected that were XlCDK2-dependent. These were all cdc2 mutants altered only in their G2/M function. Therefore XlCDK2 can influence both the G1/S and G2/M transition points of cdc2 in S. pombe.
Animals↗
I-A mutation resulted in a selective loss of an antigen-specific Ir gene function.
The immune responses to several antigens were compared in the I-A mutant mouse strain B6.C-H-2bm12 and the wild-type strain C57BL/6. With a lymph node cell proliferation assay, the response to two of these antigens, beef insulin and (TG)A-L, was demonstrated to be controlled by a gene in the I-Ab region. B6.C-H-2bm12 mice failed to respond to beef insulin, while their responses to (TG)A-L, DNP-OVA and PPD were comparable with those of the wild-type strain C57BL/6. Taken together with previous studies, these data suggest that the product of a single pleiotropic I-A gene, an Ia molecule, functions as a histocompatibility, Ia, and MLR antigen, as well as a necessary component for Ir gene function. Furthermore, the data reported here demonstrate that Ia molecules have multiple functional "Ir determinants," one of which has been altered in the B6.C-H-2bm12 mutant. The B6.C-H-2bm12 mice, therefore, represent a powerful analytical tool for the understanding of the cellular and molecular basis for Ir gene control of the immune response.
Alleles↗
CRISPR as a Tool to Uncover Gene Function in Polycystic Ovary Syndrome: A Literature Review of Experimental Models Targeting Ovarian and Metabolic Genes.
Polycystic ovary syndrome (PCOS) is a complex disorder characterized by reproductive abnormalities such as hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology, and is frequently accompanied by metabolic disturbances such as insulin resistance, obesity and dyslipidemia. Genome-wide association studies (GWASs) have identified several susceptibility loci, yet little is known about their functional implications. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has emerged as a powerful gene editing tool in bridging this gap by allowing researchers to directly target candidate genes in ovarian and metabolic pathways. For instance, experimental models have highlighted the role of CYP17A1 and DENND1A.V2 in androgen excess, anti-Müllerian hormone (AMH) in follicular arrest, and insulin receptor substrate 1 (IRS1) and PPARγ in insulin signaling and adipogenesis. To highlight the multifactorial nature of PCOS, animal models, including zebrafish and rodents, have been used to reveal interactions between reproductive and metabolic phenotypes. Nevertheless, most studies remain restricted to single-gene models, and dual-gene models or combined gene editing and hormonal induction models remain underexplored. Future research integrating precision editing, multi-omic platforms, and patient-derived organoids may provide more accurate disease models and novel therapeutic strategies.
Polycystic Ovary Syndrome↗
hairy gene function in the Drosophila eye: normal expression is dispensable but ectopic expression alters cell fates.
The regulatory gene hairy is expressed and required during early embryogenesis to control segmentation gene expression properly and during larval and pupal development to control the pattern of certain adult sensory structures. We have found the hairy protein to be expressed transiently during two stages of eye imaginal disc development, including all cells immediately anterior to the morphogenetic furrow that traverses the developing eye disc, and again in the presumptive R7 photoreceptor cells of the developing ommatidia. This pattern is conserved in a significantly diverged Drosophila species. We show that, surprisingly, ommatidia formed by homozygous hairy- mutant clones are apparently normal, indicating that hairy function in the eye is dispensable. However, we do find that ectopic expression of hairy causes numerous structural abnormalities and the alteration of cell fates. Thus, proper regulation of hairy is still essential for normal eye development. We suggest that the loss of hairy function may be compensated by other regulatory proteins, as has been observed previously for several structurally and functionally related genes involved in sensory organ development. The effects of ectopic hairy expression may result from interactions with proneural genes involved in the development of the eye and other sensory organs.
Animals↗
Immune response gene function correlates with the expression of an Ia antigen. II. A quantitative deficiency in Ae:E alpha complex expression causes a corresponding defect in antigen-presenting cell function.
A series of experiments were performed to explore the role of complementing major histocompatability complex (MHC)-linked immune response Ir genes in the murine T cell proliferative response to the globular protein antigen pigeon cytochrome c. The functional equivalence of I-E-subregion-encoded, structurally homologous E(a) chains from different haplotypes bearing the serologic specificity Ia.7 was demonstrated by the complementation for high responsiveness to pigeon cytochrome c of F(1) hybrids between low responder B 10.A(4R) (I-A (k)) or B 10.S (I-A(8)) mice and four low responder E(a)- bearing haplotypes. Moreover, this Ir gene function correlated directly with both the ability of antigen-pulsed spleen cells from these same F(1) strains to stimulate pigeon cytochrome c-primed T cells from B10.A or B10.S(9R) mice, and with the cell surface expression of the two-chain Ia antigenic complex, A(e):E(a), bearing the conformational or combinatorial determinant recognized by the monoclonal anti-Ia antibody, Y-17. The B 10.PL strain (H-2(u)), which expresses an Ia.7-positive I-E- subregion-encoded E(a) chain, failed to complement with B10.A(4R) or B10.S mice in the response to pigeon cytochrome c. However, (B10.A(4R) x B10.PL)F(1) and (B10.S x B10.PL)F(1) mice do express A(k)(e):E(u)(a) and A(8)(e):E(u)(a) on their cell surface, although in reduced amounts relative to A(k,s)(e):E(k,d,p,r)(a) complexes found in corresponding F(1) strains. This quantitative difference in Ia antigen expression correlated with a difference in the ability to present pigeon cytochrome c to B 10.A and B 10.S(9R) long-term T cell lines. Thus, (B10.A(4R) x B10.PL)F(1) spleen cells required a 10-fold higher antigen dose to induce the same stimulation as (B10.A(4R) x B10.D2)F(1) spleen cells. In addition, the monoclonal antibody, Y-17, which reacts with A(e):E(a) molecules of several strains, had a greater inhibitory effect on the proliferative response to pigeon cytochrome c of B10.A T cells in the presence of (B10.A(4R) X B10.PL)F(1) spleen cells than in the presence of (B10.A(4R) X B10.D2)F(1) spleen cells. These functional data, in concert with the biochemical and serological data in the accompanying report, are consistent with the molecular model for Ir gene complementation in which appropriate two-chain Ia molecules function at the antigen-presenting cell (APC) surface as restriction elements. Moreover, they clearly demonstrate that the magnitude of the T cell proliferative response is a function of both the concentration of nominal antigen and of the amount of Ia antigen expressed on the APC. Finally, the direct correlation of a quantitative deficiency in cell surface expression of an Ia antigen with a corresponding relative defect in antigen-presenting function provides strong independent evidence that the I-region-encoded Ia antigens are the products of the MHC-linked Ir genes.
Animals↗
Transgenic mice selectively lacking MHC class II (I-E) antigen expression on B cells: an in vivo approach to investigate Ia gene function.
The E alpha MHC class II gene with 1.4 kb of 5'-flanking and 0.5 kb of 3'-flanking sequences was introduced into (H-2b X s)F2 mice, which do not express their endogenous E alpha gene. The transgene was expressed in thymic tissue and in adherent spleen cells and was induced in peritoneal exudate cells by gamma-interferon. In contrast to the normal E alpha gene, there was no expression in B lymphocytes. Since transgenic animals made with constructs containing 3.2 kb and 2 kb of 5'-flanking sequences show normal expression pattern of the E alpha gene, it appears that deletion of 5'-flanking sequences between -1.4 kb and -2 kb inactivated or eliminated regulatory sequences required for expression of E alpha specifically in B cells. The presence of pBR327 DNA linked to the -1.4 kb E alpha transgene suppresses expression in peripheral adherent cells, yielding mice expressing E alpha only in the thymus. These mice appear to be tolerant to I-E, as measured in mixed leukocyte response experiments.
Animals↗
Characterization of the human parathyroid hormone-like peptide gene. Functional and evolutionary aspects.
The single-copy gene coding for the human parathyroid hormone-like peptide was isolated from a human placental genomic library. The gene spans 13 kilobases and contains seven exons. Exons I and II encode 5'-noncoding regions; each has its own transcription initiation site, and the two promoters are separated by over 1000 base pairs of genomic DNA. Exon III encodes the prepro-coding region, and exon IV encodes the mature peptide sequence. At the end of exon IV the splice site interrupts codon 139 of the mature peptide. Exon V, which is contiguous with exon IV, encodes a stop codon and a 3'-noncoding region. Exon VI encodes 34 additional amino acids, a stop codon, and a second 3'-noncoding region. Exon VII encodes two extra amino acids, a stop codon, and a third 3'-noncoding region. This genomic organization reveals how the multiple human parathyroid hormone-like peptide RNA transcripts, which have been observed, arise by both alternative splicing out of exons and use of multiple promoters. The mRNAs, which can potentially be formed from the primary transcript of this gene, could have one of three different carboxyl-terminal coding regions. The use of different exons to encode the different functional domains, 5'-noncoding region, pre-pro-coding region, and mature peptide region is identical to the organization of the human parathyroid hormone gene. This strongly suggests a common evolutionary origin of the two genes.
Amino Acid Sequence↗
Insights into immune-response gene function using an Ia mutant mouse strain.
The class II genes of the major histocompatibility complex (MHC) encode the immune response-associated or Ia antigens. These highly polymorphic glycoprotein heterodimers have been shown to play a pivotal role in diverse immunological functions. Although the precise molecular and cellular interactions have yet to be elucidated, structural determinations on Ia molecules are known to control the immune response to foreign antigens. Several recent advances into our understanding of how Ia molecules function were made possible by the discovery of the B6,C-H-2bm12 (bm12) mouse strain. The bm12 strain carries a mutation that occurred spontaneously in one of its genes encoding an Ia polypeptide chain. Information from studies using bm12 include (1) insights into the evolution of class II genes and its functional significance; (2) the definition of multiple functional sites on Ia and their relative importance; and (3) the demonstration that Ia conformation is important in determining its functional capacity. These findings using the bm12 mutant are reviewed in the context of other recent studies of Ia recognition.
Animals↗
Control of gene function in bacteriophage T4. IV. Post-transcriptional shutoff of expression of early genes.
The selective and sequential shutoff of synthesis of early T4 proteins in bacteria infected with DNA-negative mutants is under the active control of one or more T4-induced proteins. Selective shutoff of synthesis of early T4 proteins is accompanied by a selective degradation of distinct species of T4 mRNA. We present circumstantial evidence that selective degradation of mRNA is the cause, and not the consequence, of selective termination of expression of early T4 genes. The mutation sp62 inactivates the shutoff mechanism and prevents the selective degradation of distinct species of T4 mRNA.
Amino Acids↗