Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The yellow (y) gene of Drosophila melanogaster is required for the pigmentation of larval and adult cuticle structures. The deduced y protein sequence includes two putative N-linked glycosylation sites and a putative signal peptide, suggesting that it might be a secreted molecule. Consistent with the characteristics of a secreted protein, our in vitro translation studies using RNA synthesised from the y cDNA demonstrate that the nascent y polypeptide is a preprotein that cotranslationally translocates into the endoplasmic reticulum (ER) membrane and becomes glycosylated. The N-terminal peptide is cleaved from the preprotein between the two alanine residues at positions 21 and 22, to release the final product into the lumen of the ER. Antibodies raised against the y polypeptide detect the protein starting at 13 h post-fertilization in epidermal cells and in the cuticle structures secreted by them that later become pigmented; in addition, yellow protein is detected in the cuticle structures associated with Keilin's organs. The embryonic beta-galactosidase staining pattern of a transgene, bearing a construct in which expression of the lacZ gene is driven by the y promoter, is also described and is similar to that of the y protein. Our results indicate that the y gene product is an apically secreted protein which becomes an immobilised structural component of the pigmented cuticle.
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PMID:Apical secretion and association of the Drosophila yellow gene product with developing larval cuticle structures during embryogenesis. 146 12

Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter. In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4). The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.
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PMID:In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator. 190 59

In the alpha-complementation of beta-galactosidase an N-terminal peptide fragment (alpha-peptide) of the wild-type enzyme interacts with a defective beta-galactosidase enzyme to restore capacity for subunit assembly and activity. We have used previously a random mutagenesis and screening approach to identify a pentapeptide residue tract in the alpha-peptide that was highly tolerant of residue substitution, with some mutations conferring improved function. This tract is of clear importance for alpha-peptide function but is apparently dispensable in the intact parental enzyme. To investigate this further, we selected tract mutations and placed them into intact beta-galactosidase, at the corresponding N-terminal position as in the alpha-peptide. We then tested whether such specific tract sequences conferred properties to the whole enzyme which could be predicted from the behaviour of the defective enzyme complemented with the corresponding mutant alpha-peptide. This was shown for mutations which positively or negatively affected enzyme stability. Additionally, a subset of mutations which affected complementation efficiency in vivo were predicted to affect the formation of higher-order structures in the intact protein, and this was observed experimentally. Mutations which decreased peptide complementation dramatically decreased the level of formation of multimers in the intact protein and a mutation which increased peptide complementation produced marked enhancement of multimer formation in a protein with a pre-existing impairment in higher-order structure formation. Such subtle effects are difficult to detect directly in the whole protein by randomization/selection approaches, but in the complementing peptide the role of the residues within the pentapeptide tract is effectively amplified.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein modification from mutational analysis of an autologous peptide fragment. 212 59

The L1 and L2 capsid proteins encoded by human papillomavirus types 6 and 16 (HPV-6 and HPV-16) have been synthesized in bacteria. Antisera were raised against the HPV-6 L1- and L2-beta-galactosidase fusion proteins and against an HPV-16 L1 C-terminal peptide which was 14 amino acids long. The HPV-16 L1 peptide antibodies have been shown to be highly reactive with the HPV-16 L1-beta-galactosidase protein but not against the equivalent HPV-6 L1-beta-galactosidase fusion protein. The effectiveness of these antibodies was compared with commercially available antibovine papillomavirus type 1 (BPV-1) antibodies and the results demonstrated that the anti-BPV-1 antibodies reacted well against HPV-6 L1-beta-galactosidase but not against HPV-16 L1-beta-galactosidase. In addition, the L2 portion of the HPV-6 L2-beta-galactosidase fusion protein appeared particularly immunogenic, since antibodies raised against this fusion protein were predominantly reactive with the L2 moiety. The HPV-16 L1 peptide antibodies described here will be preferred reagents for the specific detection of HPV-16 capsid antigens, which may be particularly important in early diagnosis of HPV-16 infection.
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PMID:Expression of human papillomavirus type 6 and type 16 capsid proteins in bacteria and their antigenic characterization. 282 50

Human papillomavirus type 18 (HPV-18) has recently been closely linked with human malignant cervical lesions. The early region of the genome of the related bovine papillomavirus (BPV) has been shown to be important for the production of the transformed phenotype. BPV E6 has been shown to be a transforming protein. We report the primary structure of the HPV-18 E6 open reading frame and its predicted amino acid sequence. Both E6 protein and E6-beta-galactosidase fusion protein were synthesized in bacteria. Antisera were raised against the E6-beta-galactosidase fusion protein and against an E6 N-terminal peptide which was 14 amino acids long. We show that these antisera reacted on Western blots against E6 synthesized in bacteria. The HPV E6 antigen and antibodies described here will be useful in understanding HPV expression and its association with human malignancies and may also be diagnostically useful.
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PMID:The expression of human papillomavirus type 18 E6 protein in bacteria and the production of anti-E6 antibodies. 301 29

The tolerance of bacteriophage lambda morphogenesis for C-terminal additions to the tail tube major protein subunit (the V gene product; gpV) has been investigated. A second modified copy of the lambda V gene, either within a novel phage vector itself or plasmid-borne, was expressed during phage growth. High-level substitution of wild-type gpV by modified gpV bearing a basic C-terminal peptide sequence (RRASV; a target site for cAMP-dependent protein kinase) was possible using multiple repeats of a serine-glycine (SGGG) linker sequence. Highly purified phage bearing copies of gpV-RRASV could be efficiently phosphorylated by the appropriate protein kinase, and the incorporated label was shown to migrate exclusively at the expected size in protein gels. A large tetrameric protein (beta-galactosidase) could be incorporated into active virions in at least one copy, again using a Ser-Gly linker. These studies suggest that with a suitable spacing linker and controlled levels of expression, it is likely that a wide range of protein or peptide substitutents can be fused with gpV at its C terminus and assembled as component subunits of the tail tube.
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PMID:Assembly of functional bacteriophage lambda virions incorporating C-terminal peptide or protein fusions with the major tail protein. 775 19

Annexin 1 (ANXA1) is a member of the annexin family of phospholipid- and calcium-binding proteins with a well demonstrated role in early delayed (30 min to 3 h) inhibitory feedback of glucocorticoids in the hypothalamus and pituitary gland. This study used adrenal gland tissue from ANXA1-null transgenic mice, in which a beta-galactosidase (beta-Gal) reporter gene was controlled by the ANXA1 promoter, and wild-type control mice to explore the potential role of ANXA1 in adrenal function. RT-PCR and Western blotting revealed strong expression of ANXA1 mRNA and protein in the adrenal gland. Immunofluorescence labeling of ANXA1 in wild-type and beta-Gal expression in ANXA1-null adrenals localized intense staining in the outer perimeter cell layers. Immunogold electron microscopy identified cytoplasmic and nuclear ANXA1 labeling in outer cortical cells and capsular cells. Exposure of adrenal segments in vitro to dexamethasone (0.1 mum, 3 h) caused an increase in the amount of ANXA1 in the intracellular compartment and attached to the surface of the cells. The N-terminal peptide ANXA1(Ac2-26) inhibited corticosterone release. Corticosterone release was significantly greater from ANXA1-null adrenal cells compared with wild type in response to ACTH (10 pm to 5 nm). In contrast, basal and ACTH-stimulated aldosterone release from ANXA1-null adrenal cells was not different from wild type. Morphometry studies demonstrated that ANXA1 null adrenal glands were smaller than wild-type, and the cortical/medullary area ratio was significantly reduced. These results suggest ANXA1 is a regulator of adrenocortical size and corticosterone secretion.
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PMID:Expression and externalization of annexin 1 in the adrenal gland: structure and function of the adrenal gland in annexin 1-null mutant mice. 1715 8

We describe a novel epididymis-specific cDNA named Glb1l4, which was isolated from rat epididymis by differential display of mRNAs. Glb1l4 cDNA contains 2607 nucleotides and encodes a 637-amino acid protein with 50% similarity to mouse beta-galactosidase. The gene is located on chromosome 8q13, spanning 21 exons. Northern blot analysis reveals that Glb1l4 is specifically expressed in the caput region of epididymis and upregulated by androgen. A specific polyclonal antiserum against the N-terminal peptide of GLB1L4 has been produced. Western blot analysis and immunohistochemistry assay reveal that GLB1L4 is specifically expressed in the principal cells of the caput epididymis. Interestingly, its expression peaks at Postnatal Day 45 in mRNA level and at Postnatal Day 60 in protein level while the epididymis column cells undergo differentiation. Moreover, within this very period this secretory protein is confined inside the cell with a change of subcellular distribution pattern, which implies its important roles in the cell differentiation process. Only after the epididymal epithelium differentiation is completed and the spermatozoa enter the epididymal lumen is the GLB1L4 secreted into the luminal fluid and bound on the sperm head. Our results suggest that GLB1L4 may play various roles in principal cell differentiation and sperm maturation.
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PMID:The novel epididymis-specific beta-galactosidase-like gene Glb1l4 is essential in epididymal development and sperm maturation in rats. 1909 16

Multibillion-clone libraries of phages displaying guest peptides fused to the major coat protein pVIII (landscape libraries) are a rich source of probes for proteinaceous and non-proteinaceous targets. As opposed to the pIII-type fusion phages, which display peptides as independent structural domains, the guest peptides in the pVIII-fusion phages can be structurally and functionally influenced by contiguous subunits. To decipher the impact of the locale of a guest peptide on its affinity characteristics, we constructed a library of phages carrying beta-galactosidase-binding peptide ADTFAKSMQ at the N-terminus of the pVIII protein surrounded by random amino acids. It was found that mutagenesis of amino acids 12-19 (domain C) has polar effects on target binding affinity of the displayed peptide. The phages with highest affinity are characterized by: (i) a net electrostatic charge around -1 of domain C of the mutated phages at pH 7.0; (ii) a lower radius of cylinder coaxial to alpha-helix formed by domain C; (iii) a lower higher occupied molecular orbital (HOMO) of domain C leading to a decreased formation of hydrogen bonds and (iv) positively charged surface and torsion energy of domain C, which may require a conformational transition of N-terminal peptide ADTFAKSMQ for its binding with beta-galactosidase. Influence of the guest peptide on the diversity of mutations in the neighboring landscape area was also observed.
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PMID:Mutations in fd phage major coat protein modulate affinity of the displayed peptide. 1963 13

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