Functional characterization of a chicken major histocompatibility complex class II B gene promoter

 A 0.7 kilobase (kb) DNA fragment from the 5′ flanking region of a chicken major histocompatibility complex (MHC) class II B gene was cloned into chloramphenicol acetyltransferase (CAT) reporter vectors and was transfected into a chicken macrophage cell line that expresses a low level of MHC class II antigens. Positive orientation-dependent promoter activity of the chicken DNA was evident in a reporter construct containing an SV40 enhancer. Deletion analysis of this 0.7 kb DNA fragment revealed a short fragment in the 3′ end that was crucial for the promoter function and negative regulatory elements (NRE) located further upstream. The conserved MHC class II X and Y boxes did not have a significant effect on promoter activity. Sequence analysis of the 0.7 kb class II B gene upstream region suggests possible involvement of interferon (IFN), E twenty-six specific (ETS)-related proteins, and other factors in regulating this promoter. A chicken T-cell line culture supernatant increased surface expression of MHC class II antigens, as well as class II promoter activity, in this macrophage cell line. This first functional characterization of a chicken MHC class II B gene promoter will aid in understanding the regulatory mechanisms that control the expression of these genes. Received: 9 July 1996 / Revised: 7 October 1996     

Abscisic acid-regulated Glb1 transient expression in cultured maize P3377 cells

In a study of the 5′-flanking sequence of the Zea mays L. (maize) Glb1 gene in vitro, serial promoter deletions were generated and linked with the β-glucuronidase (GUS) reporter gene. The promoter deletion-GUS fusions were introduced into the maize P3377 cell line by particle bombardment. GUS assays indicated that treatment of the maize cultured cells with abscisic acid (ABA) was required for Glb1-driven GUS transient expression, and that the –272-bp sequence of the Glb1 promoter was sufficient for ABA-regulated expression of GUS. The longest undeleted sequence used, –1391 GUS, showed relatively low expression which could be indicative of an upstream silencer element in the Glb1 promoter between –1391 and –805. Further studies show that the Glb1-driven GUS activity of bombarded maize P3377 cells increases with increasing ABA concentration (up to 100–300 μm). Site-directed mutagenesis of a putative ABA response element, Em1a, abolished GUS expression in P3377 cells. This observation indicated that the Em1a sequence in the Glb1 5′ regulatory region is responsible for the positive ABA regulation of gene expression. Received: 9 May 1997 / Revision received: 9 November 1997 / Accepted: 8 December 1997     

Somaclonal-variation-induced aluminum-sensitive mutant from an aluminum-inbred maize tolerant line

Somaclonal-variation-induced multiple mutations were observed in a progeny of the S1587 plant, regenerated from type I calli of the aluminum-tolerant inbred maize line Cat-100-6. After five generations of self-pollination, 14 progeny families of the S1587 somaclone were found to show aluminum toxicity symptoms with altered root tip morphology and reduced primary root growth. The most sensitive progeny, S1587-17, was crossed to the Cat-100-6 inbred line. The parental lines and the F1 were tested in nutrient solutions containing an aluminum activity gradient of 0–93 ⋅ 10^–6. The heterozygote behaves like the tolerant parent at aluminum activities up to 40 ⋅ 10^–6 and showed an intermediate phenotype at higher aluminum concentrations. Histological sections of aluminum-treated roots from tolerant and sensitive plants stained with hematoxylin, an aluminum marker, showed a progressive destruction of the root tip of the aluminum-sensitive genotype over time and indicated that tolerance in Cat-100-6 could be due to an aluminum exclusion mechanism. Segregation analysis of the F2 and backcross to the sensitive parent based on root morphology of plants subjected to an aluminum activity of 30 ⋅ 10^–6 showed the typical 3:1 and 1:1 tolerant:sensitive segregation ratios, respectively, indicating that tolerance in the Cat-100-6 inbred maize line is controlled by a single nuclear, semidominant gene, named Alm1. Received: 9 May 1996 / Revision received: 24 February 1997 / Accepted: 8 March 1997     

Telomeric length and telomerase activity vary with age in peripheral blood cells obtained from normal individuals

The telomerase activity and length of telomeres of peripheral blood mononuclear cells obtained from 124 healthy individuals aged 4–95years was measured. Telomerase activity level was semiquantitatively assessed by a fluorescent-telomeric repeat amplification protocol (fluorescent-TRAP) using an internal telomerase assay standard, fluorescent primers and an automated laser fluorescent DNA sequencer. Telomeric length, measured by assay of terminal restriction fragments (TRFs), was determined in HinfI-digested DNA by Southern blot analysis using a (TTAGGG)₄ probe. TRF length was determined in 80 individuals and age-related progressive reduction of size was observed. TRF length in peripheral blood mononuclear cells obtained from normal individuals (aged 4–39years) decreased by approximately 84bp per year, while in individuals aged ≥40years it decreased by 41bp per year. In contrast, telomerase activity showed an apparent biphasic pattern with aging. Individuals aged 4–39years showed a progressive decrease in telomerase activity, whereas 65% of those aged ≥40years showed relatively stable but very low telomerase activity, and the remaining individuals aged ≥40years had no detectable telomerase activity. These data obtained from normal individuals might in the future be of value to help risk stratify and manage the care of patients with leukemia. Received: 18 August 1997 / Accepted: 10 December 1997     

Expression and secretion of functional miniantibodies McPC603scFvDhlx in cell-wall-less L-form strains of Proteus mirabilis and Escherichia coli : A comparison of the synthesis capacities of L-form strains with an E. coli producer strain

The paper describes the synthesis of the phosphorylcholine-binding miniantibody McPC603scFvDhl x in cell-wall-less L-form strains of Escherichia coli and Proteus mirabilis. Cells of these strains were transformed with the plasmid pACK02scKan, carrying the miniantibody (miniAb) coding sequence under the control of the lac promoter. L-form transformants of both species were able to synthesize the functional miniAb as an extracellular soluble product. The highest quantities were obtained by P. mirabilis L-form strains after induction with 5 mM isopropyl β-d-thiogalactopyranoside (IPTG). Yields of 45–75 mg/l total antibody protein and of 10–18 mg/l functional miniAb were estimated in the growth medium of shaking cultures 40–80 h after induction with IPTG. About 10% of the active miniAb remained cell-bound. The yields of functional miniAb could be optimized by lowering the growth temperature from 37 °C to 26–32 °C and by supplementation of the medium with 80 mM sodium fumarate. A comparison of the specific activities revealed that the P. mirabilis L-form strains have a similar synthesis capacity (2–4 mg functional miniAb/g cell dry weight) to that of the producer strain E. coli RV308. The results show that the processes of correct folding and assembling of the miniAb molecules are possible without the periplasmic compartment. Received: 14 April 1997 / Received revision: 17 July 1997 / Accepted: 25 August 1997     

The differentiation-dependent inhibition of SV40 early promoter/enhancer activity in 3T3-L1 cells is mediated through promoter and not enhancer sequences

Using a plasmid bearing chloramphenicol acetyltransferase (CAT) gene controlled by Simian virus 40 (SV40) early promoter/enhancer complex (pA0cat), we analyzed functional enhancer motifs in 3T3-L1 fibroblast and adipocyte cells. Deletion mutant series of pA0 at the enhancer complex showed that gene expression both in fibroblast and adipocyte cells was dependent on a similar set of enhancer motifs. When pA0 was introduced into 3T3-L1 fibroblasts and the cells were induced to differentiate into adipocytes, CAT activity expressed in fibroblasts was suppressed. Experiments with the deletion mutants at the enhancer complex showed that the suppression was not related to any enhancer motif, and CAT activity was observed with a plasmid having only the promoter sequence. When pA0cat was co-transfected with excess of promoter sequence, the suppression in adipocytes was counteracted. This suggested that negativetrans-acting factors of the promoter sequence were responsible for the suppression in adipocytes.Abbreviations CAT chloramphenicol acetyltransferase - CAT the gene encoding CAT - SV40 Simian virus 40 - Asc-P ascorbic acid phosphate