Localization of a gene for human α-galactosidase B (=N-Acetyl-α-D-Galactosaminidase) on chromosome 22

Summary The localization of the structural gene for human -galactosidase B (=N-acetyl--galactosaminidase) was investigated by means of man-Chinese hamster and man-mouse somatic cell hybrids. The hybrid clones were analyzed for chromosomes and for a large number of known enzyme markers. The lysates of the hybrid cells were treated with Sepharose-coupled antihuman -galactosidase B and the activity of the adsorbed enzyme was measured on the Sepharose beads as N-acetyl--galactosominidase. The results show that the structural gene for human -galactosidase B is situated on chromosome 22, and that there is no structural relationship between human -galactosidase A and human -galactosidase B.     

Ecdysterone selectively stimulates the expression of a 23000-Da heat-shock protein-β-galactosidase hybrid gene in cultured Drosophila cells

To study the regulated expression of cloned heat-shock genes in homologous cells, hybrid Drosophila heat-shock-Escherichia coli β-galactosidase genes were constructed. Segments of the ecdysterone-inducible 23,000-Da heat-shock protein (hsp23) gene and of two other hsp genes (hsp84 and 70), which are not hormone regulated, were functionally linked to the bacterial coding sequence, and the resulting hybrid genes were introduced into cultured, hormone-responsive Drosophila cells by transfection. All hybrid genes directed the synthesis of E. coli-specific β-galactosidase in heat-treated cells. hsp23 hybrid gene expression was stimulated strongly by ecdysterone, while the activities of the other hybrid genes were not affected at all by the hormone. A hybrid gene with only 147 bp of hsp23 promoter sequence could not be activated by either heat or ecdysterone treatment. Thus, far upstream sequences contain signals required for the regulated expression of the hsp23 gene in Drosophila cells.     

Purification and partial characterization of β-galactosidase from Tritrichomonas foetus

The work presented in this paper describes the purification and properties of a β-galactosidase from the protozoan Tritrichomonas foetus. An inexpensive and straightforward method for extraction of the enzyme involving ammonium sulphate precipitation, ion exchange and affinity chromatography resulted in a high level of purification. After purification β-N-acetylglucosaminidase was the only enzyme present as a contaminant at a significant level. The β-galactosidase isolated had a pH optimum of 5.8. The K_m determined at pH 5.8 was found to be 2.2 mM. Interesting results were obtained when studies were carried out to determine the effect of various metal ions on enzyme activity. Of the metal ions used in this study only manganese ions were found to activate the enzyme. This seems to be a characteristic of trichomonad enzymes, as N-acetyl-β-glucosaminidase, a-galactosidase and N-acetyl-a-galactosaminidase are also activated by manganese ions. The strongest inhibition was recorded with lead and to a lesser extent by zinc. The result with lead is not unexpected as the heavy metal is known to cause irreversible inhibition by binding to the amino-acid backbone of the enzyme. The result with zinc is interesting as high levels of zinc are present and trichomonads are known to be apathogenic in semen. The purified β-galactosidase was found to have the capacity to hydrolyse lactose (Gal β1-4 Glc), lacto-N-biose 1 (Gal β1-3 GlcNAc) and N-acetyllactosamine (Gal β1-4 GlcNAc). When the enzyme was applied to a non-denaturing polyacrylamide gel a single band was observed when stained with Coomassie brilliant blue. This band coincided with that obtained when the gel was stained with p-nitrophenyl β-galactopyranoside. When the same gel was incubated with p-nitrophenyl N-acetyl β-glucopyranoside a band was detected which did not coincide with that of β-galactosidase. Since the β-N-acetylglucosaminidase enzyme does not move to the same position on a non-denaturing gel as the β-galactosidase, we will use this technique to isolate the latter enzyme and determine the N-terminal sequence as a prelude to cloning and further study of the gene. This revised version was published online in November 2006 with corrections to the Cover Date.     

Estimation of DNA Base Composition by High Performance Liquid Chromatography of Its Nuclease PI Hydrolysate

The Saccharomyces diastaticus glucoamylase encoded by ST A1 contains two signal sequences for potent secretion of the enzyme, a hydrophobic leader peptide (HL), and a tract consisting of threonine- and serine-rich sequences (TS); hybrid proteins of Escherichia coli β-galactosidase carrying both HL and TS are secreted through the cytoplasmic membrane to the cell-surface fraction of yeast cells, but those carrying either HL or TS are not. To investigate the molecular mechanisms for these signal sequences, we have isolated a dominant mutation, SSD1, which suppresses a secretory defect caused by deletion of these sequences. Yeast cells harboring the mutation secreted hybrid β-galactosidase proteins carrying either HL or TS into the cell-surface fraction. Even β-ga!actosidase itself was secreted to the cell surface in the mutant. These results suggest that HL and TS interact with a wild- type ssd1⁺ gene product to promote protein secretion.     

A T4 DNA fragment containing genes for the baseplate central plug: Endonuclease restriction, gene expression and cell wall changes

Summary A fragment of Escherichia coli bacteriophage T4D DNA, containing 6.1 Kbp which included the six genes (genes 25, 26, 51, 27, 28 and 29) coding for the tail baseplate central plug has been partially characterized. This DNA fragment was obtained originally by Wilson et al. (1977) by the action of the restriction enzyme EcoRI on a modified form of T4 DNA and was inserted in the pBR322 plasmid and then incorporated into an E. coli K12 strain called RRI. This plasmid containing the phage DNA fragment has now been reisolated and screened for cleavage sites for various restriction endonucleases. Restriction enzymes Bgl 11 and Xbal each attacked one restriction site and the enzyme Hpa 1 attacked two restriction sites on this fragment. The combined digestion of the hybrid plasmid containing the T4 EcoRI DNA fragment conjugated to the pBR322 plasmid with one of these enzymes plus Bam H1 restriction enzyme resulted in the localization of the restriction site for Bgl 11, Xba 1 and Hpa 1. Escherichia coli strain B cells were transformed with this hybrid plasmid and found to have some unexpected properties. E. coli B cells, which are normally restrictive for T4 amber mutants and for T4 temperature sensitive mutants (at 44°) after transformation, were permissive for 25am, 26am and 26^Ts, 51am, and 51^Ts, 27^Ts, and 28^Ts T4 mutants. Extracts from the transformed E. coli cells were found in complementation experiments to contain the gene 29 product, as well as the gene 26 product, the gene 51 product, and the gene 27 product. The complementation experiments and the permissiveness of the transformed E. coli B cells to the various conditional lethal mutants clearly showed that the six T4 genes were producing all six gene products in these transformed cells. However, these cells were not permissive for T4 amber mutants in genes 27, 28, and 29. The transformed E. coli B cells, as compared to untransformed cells, were found to have altered outer cell walls which made them highly labile to osmotic shock and to an increased rate of killing by wild type T4 and all T4 amber mutants except for T4 am29. The change in cell walls of the transformed cells has been found to be due to the T4 baseplate genes on the hybrid plasmid, since E. coli B transformed by the pBR322 plasmid alone does not show the increase in osmotic sensitivity.     

Residual activity of α-galactosidase A in Fabry's disease

The α-galactosidase A activity from fibroblasts of five Fabry patients and five controls has been separated from α-galactosidase B through small DEAE-cellulose columns and in some experiments by treatment of the fibroblast extracts with Sepharose coupled to anti-α-galactosidase B antibodies. By these independent methods, it has been shown that there is a residual α-galactosidase A in Fabry's disease, which is immunologically similar to the α-galactosidase A from the controls. The α-galactosidase A from all of the patients and controls has the same apparent K_m value for the synthetic substrate 4-methylumbelliferyl-α-galactoside. Four out of five patients have a thermostable α-galactosidase A, while the fifth has a thermolabile enzyme like that from the controls. The amount of immunologically active α-galactosidase A seems to be decreased in the patients tested.     

Murine CD8+ T cells but not macrophages express the vitamin D 1α-hydroxylase

The active form of vitamin D, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] is synthesized by the 1α-hydroxylase, which is encoded by the Cyp27B1 gene. Using transgenic mice that have replaced the Cyp27B1 gene with the bacterial lacZ reporter gene (β-galactosidase), the inflammatory conditions that induce Cyp27B1 in the immune system were probed. A variety of stimuli including lipopolysaccharide, anti-CD3 or PMA/ionomycin were used to stimulate splenocytes and bone marrow derived macrophage in vitro. Only anti-CD3 stimulation resulted in a low induction of β-galactosidase activity in the spleen, indicating that T cells might be a source of Cyp27B1. In vivo, challenge with lipopolysaccharide, α-galactosylceramide, and Listeria monocytogenes failed to induce β-galactosidase activity outside of the kidneys. During more prolonged and severe inflammation there was staining in both the lungs and the gastrointestinal tract for β-galactosidase. Furthermore, wild-type reconstitution of the hematopoietic cell population in Cyp27B1 KO mice protected the mice from experimental colitis. T cell production of Cyp27B1 activity was shown to be from the CD8+ but not the CD4+ T cell population. CD8+ T cells expressed the reporter gene only after 48 h of stimulation. The data is consistent with a model where CD8+ T cells are activated to produce Cyp27B1 and 1,25(OH)₂D₃ that serves to turn off the local immune response.     

Cell specificity in the developmental regulation of acid hydrolases by temporal genes

The cell specificity of expression of three distinct trans acting temporal gene systems determining the developmental control of α-galactosidase, β-galactosidase and β-glucuronidase was tested in mouse liver. For α-galactosidase and β-galactosidase, expression was limited to hepatocytes; no effect was seen in nonhepatocytes. For β-glucuronidase the data suggest that expression of the Gus-t temporal locus is also limited to hepatocytes, and that the smaller enzyme reduction seen in nonhepatocytes of some strains is due to a separate systemic regulatory locus that is also present in the [Gus] gene complex. We conclude that the temporal gene-determined timing mechanisms initiating switches in rates of enzyme synthesis are intrinsic to the cells themselves and are not communicated to adjacent cells. This conclusion applies to the temporal locus for β-glucuronidase that is proximate to its structural gene as well as those for α-galactosidase and β-galactosidase that are distant from the structural genes that they regulate.     

Reporter genes in transgenic mice

Althoughin vivo models utilizing endogenous reporter genes have been exploited for many years, the use of reporter transgenes to dissect biological issues in transgenic animals has been a relatively recent development. These transgenes are often, but not always, of prokaryotic origin and encode products not normally associated with eukaryotic cells and tissues. Some encode enzymes whose activities are detected in cell and tissue homogenates, whereas others encode products that can be detectedin situ at the single cell level. Reporter genes have been used to identify regulatory elements that are important for tissue-specific gene expression or for development; they have been used to producein vivo models of cancer; they have been employed for the study ofin vivo mutagenesis; and they have been used as a tool in lineage analysis and for marking cells in transplanation experiments. The most commonly usedin situ reporter gene islacZ, which encodes a bacterial -galactosidase, a sensitive histochemical marker. Although it has been used with striking success in cultured cells and in transgenic mouse embryos, its postnatalin vivo expression has been unreliable and disappointing. Nevertheless, the ability to express reporter genes in transgenic mice has been an invaluable resource, providing insights intoin vivo biological mechanisms. The development of newin vivo models, such as those in which expression of transgenes can be activated or repressed, should produce transgenic animal systems that extend our capacity to address heretofore unresolved biological questions.     

Structure and expression of the Lyt-3 agene of C.AKR mice

The mouse Lyt-3 ^agene, which encodes the Lyt-3.1 T-cell surface alloantigen of the C.AKR strain, has been cloned, and the nucleotide sequence of its exons and more than 2 kb of 5 flanking sequence have been determined. The gene extends over approximately 16 kb of DNA and consists of six exons encoding leader, leader plus V-like domain, membrane-proximal, transmembrane, and cytoplasmic domains. The only difference between the coding region of the Lyt-3 ^agene and the cDNA sequences reported for Lyt-3 ^b(Nakauchi et al. 1987, Panaccio et al. 1987) is at position 77 of the mature protein where Lyt-3 ^aencodes serine and Lyt-3 ^bencodes arginine. This substitution must therefore be the basis for the serological distinction between the Lyt-3.1 and Lyt-3.2 alloantigens. Potential TATA and CAAT sequences, two Sp1 protein binding sites, two extended repeats of the dinucleotide, CA, a number of short inverted repeats, and an inverted segment of the mouse B1 repetitive sequence are found 5 to the Lyt-3 ^agene. Two consensus poly-A addition signals and a complete copy of the mouse B1 sequence are found 3 to the gene. Both B1-related regions are flanked by short direct repeats suggesting that they arose by an insertional mechanism. Cotransfection of the Lyt-3 ^agene together with a cloned Lyt-2 ^agene resulted in expression of both Lyt-2 and Lyt-3.1 on the surface of Ltk^– and BW5147 cells. Transfection of the Lyt-3 ^agene without Lyt-2 ^aled to expression of Lyt-3-related cellular RNA but did not result in surface expression of Lyt-3.1, suggesting that the Lyt-3 glycoprotein is not expressed on the cell surface in the absence of Lyt-2.     

Scanning assay of β-galactosidase activity

β-galactosidase, encoded by the lacZ gene in E. coli, can cleave lactose and structurally related compounds to galactose and glucose or structurally related products. Its activity can be measured using an artificial substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG). Miller firstly described the standard quantitative assay of β-galactosidase activity in the cells of bacterial cultures by disrupting the cell membrane with the permeabilization solution instead of preparing cell extracts. Therefore, β-galactosidase became one of the most widely used reporters of gene expression in molecular biology to reflect intracellular gene expression difference. But the Miller assay procedure could not monitor the β-galactosidase reaction in real time and its results were greatly influenced by some operations in the Miller procedure, such as permeabilization time, reaction time and concentration of the cell suspension. A scanning method based on the Miller method to determine the intracellular β-galactosidase activity in E. coli Tuner (DE3) expressing β-galactosidase in real time was developed and the permeabilization time of cells was optimized for that. The comparison of 3 assays of β-galactosidase activity (Miller, colorimetric and scanning) was made. The results proved that scanning method for the determination of enzyme activity with using ONPG as substrate is simple, fast and reproducible.     

High levels of inducible expression of cloned β-galactosidase ofKluyveromyces lactis inSaccharomyces cerevisiae

Summary The LAC4 gene ofKluyveromyces lactis CBS2360 coding for -galactosidase was isolated from aK. lactis gene bank. The gene was when cloned in a yeast expression vector pBCL26, derived from pLG2 (Guarente 1983), under the control of the inducible GAL1-10 USA/CYC1 yeast hybrid promoter. Two constructions were obtained, pBCLG2 and pBCLG4, that bear the LAC4 gene in the two opposite orientations and we have studied the expression ofK. lactis -galactosidase in yeast cells transformed with these two plasmids and under different growth conditions. High levels of expression induced by galactose were observed with pBCLG2, which bears the LAC4 gene in the correct orientation, while a low constitutive level of expression was observed in pBCLG4 transformants both in glucose and in galactose media. The expression of the heterologous protein, under induced conditions, appears to be strongly influenced by the growth phase of the culture, with a sharp increase of the specific activity of the enzyme and of its level, calculated as percent of total protein, at the beginning of the stationary phase of growth, during which time the -galactosidase reaches a level of 15% of total cellular protein.     

Molecular Characterization and Therapeutic Potential of a Marine Bacterium Pseudoalteromonas sp. KMM 701 α-Galactosidase

An α-galactosidase capable of converting B red blood cells into the universal blood type cells at the neutral pH was produced by a novel obligate marine bacterium strain KMM 701 (VKM B-2135 D). The organism is heterotrophic, aerobic, and halophilic and requires Na⁺ ions and temperature up to 34°C for its growth. The strain has a unique combination of polysaccharide-degrading enzymes. Its single intracellular α-galactosidase exceeded other glycoside hydrolases in the level of expression up to 20-fold. The α-galactosidase was purified to determine the N-terminal amino acid sequences and new activities. It was found to inhibit Corynebacterium diphtheria adhesion to host buccal epithelium cell surfaces with high effectiveness. The nucleotide sequence of the homodimeric α-galactosidase indicates that its subunit is composed of 710 amino acid residues with a calculated Mr of 80,055. This α-galactosidase shares structural property with 36 family glycoside hydrolases. The properties of the enzyme are likely to be highly beneficial for medicinal purposes.     

A new mouse cell-surface antigen (Ly-m18) defined by a monoclonal antibody

Spleen cells from C3H/An mice immunized with spleen cells of C57BL/6-H-2 ^k mice were fused with myeloma cell line NS.1. One established hybrid cell line continuously secreted antibody that recognized a new surface antigen provisionally called Ly-m18. The new alloantigen is expressed on 90 percent of thymus cells, 55 percent of spleen cells, and 45 percent of either lymphnode or bone-marrow cells. It is also expressed on cells derived from brain, kidney, and liver. Fifty percent of either peripheral T or B cells express the Ly-m18 antigen, and some tumor cell lines with T, B, pre-B or stem cell characteristics are Ly-m18 (+). The strain distribution pattern distinguishes Ly-m1 8 antigen from all other murine lymphocyte alloantigens. The typing data of two sets of CXB and AKXL recombinant inbred strains indicate that the Ly-m18 gene is linked to the Ltw-2 locus which has not yet been assigned to a chromosome.Abbreviations used in this paper RI recombinant inbred - Con-A concanavalin A - LPS lipopolysaccharide - MLR mixed lymphocyte reaction The prefix m (monoclonal) is used following a suggestion by Klein and co-workers (1979).     

Functional Identification of a Putative β-Galactosidase Gene in the Special lac Gene Cluster of Lactobacillus acidophilus

The putative β-galactosidase gene (lacZ) of Lactobacillus acidophilus has a very low degree of homology to the Escherichia coli β-galactosidase gene (lacZ) and locates in a special lac gene cluster which contains two β-galactosidase genes. No functional characteristic of the putative β-galactosidase has been described so far. In this study, the lacZ gene of L. acidophilus was hetero-expressed in E. coli and the recombinant protein was purified by a three-step procedure. The product of the lacZ gene was also extracted from L. acidophilus ATCC 4356 and active staining was carried out. The enzymatic properties of the purified recombinant LacZ were assayed. The results of hetero-expression showed the recombinant LacZ without tag had β-galactosidase activity. The purified recombinant LacZ had a specific activity of 43.2 U/mg protein. The result of active staining showed that the functional product of the lacZ gene did exist in L. acidophilus. The L. acidophilus β-galactosidase (LacZ) had an optimal pH of 6, an optimal temperature of 37°C and could hydrolyze 73% of lactose in milk in 30 h at 10°C. The L. acidophilus β-galactosidase (LacZ) was identified as cold-adapted β-galactosidase in this study for the first time, and may be useful for lactose removal from dairy products at low temperatures.     

Effects of estradiol-17β in the male xenopus laevis: Isolation and translation of cytoplasmic messenger rna populations

Summary Total Xenopus liver cytoplasmic RNA isolated following long-term estrogen administration (14 days) was fractioned using Sepharose 4B chromatography. One of the Sepharose 4B peaks was shown to contain RNA with a molecular weight reported for vitellogenin mRNA (34S). The presence of estrogeninduced vitellogenin mRNA in the peak 5 RNA was determined by translation of the RNA in the oocyte and analysis of the oocyte translational products by immunoprecipitation with anti-vitellogenin.Sepharose 4B peaks 2 and 3 were also observed to contain estrogen induced mRNA populations sedimenting between 9-18S. These findings suggest that Sepharose 4B chromatography might prove useful in separating different mRNA populations following estrogen-induced gene activation.     

Comparision of the methods available for the purification of hybrid β-galactosidase proteins produced under the control of an anaerobically-induced promoter

Summary Various methods available for purifying -galactosidase fusion proteins were compared in an attempt to purify a hydrophobic hybrid protein, NirC' 'LacZ, produced under the control of an anaerobically-induced promoter. Conventional ion-exchange techniques and affinity chromatography on p-aminobenzyl-thiogalactoside Sepharose CL-4B, supplied by Sigma Chemical Co., were unsatisfactory. In contrast, immunoaffinity chromatography on anti--galactosidase Protosorb^TM, obtained from Promega Biotech, or with immnunoadsorbents prepared in our laboratory, produced a purified hybrid protein which was suitable for N-terminal amino acid analysis.     

Restoration of Haemolytic Complement Activity in C5-deficient Mice by Gene Complementation in Hybrid Cells

THE fusion of cells by inactivated Sendai virus provides a method for the study of gene regulation in mammalian cells. Two cells, each with a specific enzyme deficiency, have been joined to yield a hybrid cell containing both previously deficient enzymes^{1, 2}. Harris and his co-workers have shown that terminally differentiated chick erythrocyte nuclei, when introduced into HeLa cytoplasm by cell fusion, are “reactivated” and direct the elaboration, by the hybrid cell, of chick proteins^{3, 4}. These in vitro examples of gene complementation in hybrid cells give no indication of the functional significance of the elaborated gene products. We have used the cell fusion technique to develop an experimental model for the potential-correction of a specific genetic defect in a living animal.