Cloning of the Zea mays controlling element Ac from the wx-m7 allele

Summary The cloning of the controlling element Ac from the wx-m7 allele of Zea mays is described. The cloned fragment carries a 4.3 kb insertion that by restriction analysis is indistinguishable from the Ac insertion in Ac wx-m9. It is located approximately 2.5 kb upstream of the Ac wx-m9 insertion. Offprint requests to: P. Starlinger     

Ac transposition in transgenic tomato plants

Summary As an initial step towards developing a transposon mutagenesis system in tomato, the maize transposable element Ac was transformed into tomato plants via Agrobacterium tumefaciens. Southern analysis of leaf tissue indicated that in nine out of eleven transgenic plants, Ac excised from the T-DNA and reintegrated into new chromosomal locations. The comparison of Ac banding pattern in different leaves of the same primary transformant provided evidnece for transposition during later stages of transgenic plant development. There was no evidence of Ds mobilization in tomato transformants.     

Role of the Blood-Brain Barrier in the Formation of Long-Chain ω-3 and ω-6 Fatty Acids from Essential Fatty Acid Precursors

Elongated, more highly polyunsaturated derivatives of linoleic acid (18:2 omega-6) and linolenic acid (18:3 omega-3) accumulate in brain, but their sites of synthesis and mechanism of entry are not well characterized. To investigate the role of the blood-brain barrier in this process, cultured murine cerebromicrovascular endothelia were incubated with [1-14C]18:2 omega-6 or [1-14C]18:3 omega-3 and their elongation/desaturation products determined. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary product from 18:3 omega-3 was 20:5 omega-3. Although these products were found primarily in cell lipids, they were also released from the cells and gradually accumulated in the extracellular fluid. Eicosanoid production was observed from the 20:4 omega-6 and 20:5 omega-3 that were formed. No 22:5 omega-6 or 22:6 omega-3 fatty acids were detected, suggesting that these endothelial cells are not the site of the final desaturation step. Although the uptake of 18:3 omega-3 and 18:2 omega-6 was nearly identical, 18:3 omega-3 was more extensively elongated and desaturated. Competition experiments demonstrated a preference for 18:3 omega-3 by the elongation/desaturation pathway. These findings suggest that the blood-brain barrier can play an important role in the elongation and desaturation of omega-3 and omega-6 essential fatty acids during their transfer from the circulation into the brain.     

Pulmonary microcirculatory kinetics of neutrophils deficient in leukocyte adhesion-promoting glycoproteins

The mechanism that causes neutrophils to sequester in the pulmonary circulation is unknown. Because the CD11/CD18 glycoprotein family on the surface membrane of neutrophils participates in many adhesive interactions with the endothelium, we investigated the role of these proteins in the intravascular sequestration of pulmonary neutrophils. Neutrophils were isolated from normal dogs and from the only living dog known to have leukocyte adhesion deficiency disease, an inherited deficiency of the CD11/CD18 adhesion family. The neutrophils were labeled with fluorescein dye, injected into normal recipient dogs, and their passage through the pulmonary microcirculation was recorded by in vivo videofluorescence microscopy through a transparent thoracic window. Transit times for normal and deficient neutrophils were similar over a wide range of hemo-dynamic conditions. Activation by zymosan-activated plasma, which increases the surface membrane expression of CD11/CD18, prolonged the transit of normal neutrophils but did not alter the transit time of the deficient neutrophils. These results indicate that neutrophil CD11/CD18 adhesion-promoting glycoproteins are not involved in the normal pulmonary sequestration of neutrophils but have a significant role in the arrest of activated neutrophils in the pulmonary capillaries.     

The Refined Three-Dimensional Structure of Pectate Lyase E from Erwinia chrysanthemi at 2.2 A Resolution

The crystal structure of pectate lyase E (PelE; EC 4.2.2.2) from the enterobacteria Erwinia chrysanthemi has been refined by molecular dynamics techniques to a resolution of 2.2 A and an R factor (an agreement factor between observed structure factor amplitudes) of 16.1%. The final model consists of all 355 amino acids and 157 water molecules. The root-mean-square deviation from ideality is 0.009 A for bond lengths and 1.721[deg] for bond angles. The structure of PelE bound to a lanthanum ion, which inhibits the enzymatic activity, has also been refined and compared to the metal-free protein. In addition, the structures of pectate lyase C (PelC) in the presence and absence of a lutetium ion have been refined further using an improved algorithm for identifying waters and other solvent molecules. The two putative active site regions of PelE have been compared to those in the refined structure of PelC. The analysis of the atomic details of PelE and PelC in the presence and absence of lanthanide ions provides insight into the enzymatic mechanism of pectate lyases.     

Parallel β-domains: a new fold in protein structures

A new type of structural domain, composed of parallel β-strands folded into a coiled structure, has been observed in several protein structures within the past year. An analysis of the basic motif indicates that there are two distinct types, with variations likely to be discovered in the future.     

Food and water resources used by the Madagascan hissing-cockroach mite,Gromphadorholaelaps schaeferi

We determined the food source and water balance properties of the hissing-cockroach mite, Gromphadorholaelaps schaeferi. The food source for mites was identified using Evans blue dye by direct injection into a fasting host cockroach, Gromphadorhina portentosa, or by incorporation into cockroach food. No coloration was observed in mites on dye-injected cockroaches, but coloration was present in mites when only the food for the cockroaches had been stained. Thus, the mites are scavengers of cockroach food, and are not parasitic as previously thought. Our results demonstrate that the mites can absorb water from the air anywhere between 0.84 and 0.93 a _v (%RH/100), and wax-block experiments revealed that the mouth is the site of uptake. The mites are normally clumped together on the host, typically in between the cockroach's legs and around the spiracles. Water loss rates for mites in groups (0.16% h^-1) were far lower than for isolated mites (0.30% h^-1), suggesting a group effect with regard to water balance. Above the transition temperature of 30°C rate of water loss was rapid. The sites occupied by mites on the cockroach's body seem to be highly specific for feeding and absorption of water vapour.     

Identification of domains within the human cytomegalovirus major immediate-early 86-kilodalton protein and the retinoblastoma protein required for physical and functional interaction with each other.

The human cytomegalovirus major immediate-early 86-kDa protein (IE2 86) plays an important role in the trans activation and regulation of HCMV gene expression. Previously, we demonstrated that IE2 86 contains three regions (amino acids [aa] 86 to 135, 136 to 290, and 291 to 364) that can independently bind to in vitro-translated Rb when IE2 86 is produced as a glutathione S-transferase fusion protein (M. H. Sommer, A. L. Scully, and D. H. Spector, J. Virol. 68:6223-6231, 1994). In this report, we have elucidated the regions of Rb involved in binding to IE2 86 and have further analyzed the functional nature of the interaction between these two proteins. We find that two domains on Rb, the A/B pocket and the carboxy terminus, can each independently form a complex with IE2 86. In functional assays, we demonstrate that IE2 86 and another IE protein, IE1 72, can counter the enlarged flat cell phenotype, but not the G1/S block, which results from expression of wild-type Rb in the human osteosarcoma cell line Saos-2. Mutational analysis reveals that there are two domains on IE2 86 that can independently affect Rb function. One region (aa 241 to 369) includes the major Rb-binding domain, while the second maps to the amino-terminal region (aa 1 to 85) common to both IE2 86 and IE1 72. These data show that Rb and IE2 86 physically and functionally interact with each other via at least two separate domains and provide further support for the hypothesis that IE2 86 may exert its pleiotropic effects through the formation of multimeric protein complexes.