Relationship between human immunodeficiency virus type 1 Gag multimerization and membrane binding.

The human immunodeficiency virus type 1 (HIV-1) Gag precursor, Pr55(Gag), is necessary and sufficient for the assembly and release of viruslike particles. Binding of Gag to membrane and Gag multimerization are both essential steps in virus assembly, yet the domains responsible for these events have not been fully defined. In addition, the relationship between membrane binding and Gag-Gag interaction remains to be elucidated. To investigate these issues, we analyzed, in vivo, the membrane-binding and assembly properties of a series of C-terminally truncated Gag mutants. Pr55(Gag) was truncated at the C terminus of matrix (MAstop), between the N- and C-terminal domains of capsid (CA146stop), at the C terminus of capsid (p41stop), at the C terminus of p2 (p43stop), and after the N-terminal 35 amino acids of nucleocapsid (NC35stop). The ability of these truncated Gag molecules to assemble and release viruslike particles and their capacity to copackage into particles when coexpressed with full-length Gag were determined. We demonstrate that the amount of truncated Gag incorporated into particles is incrementally increased by extension from CA146 to NC35, suggesting that multiple sites in this region are involved in Gag multimerization. Using membrane flotation centrifugation, we observe that MA shows significantly reduced membrane binding relative to full-length Gag but that CA146 displays steady-state membrane-binding properties comparable to those of Pr55(Gag). The finding that the CA146 mutant, which contains only matrix and the N-terminal domain of capsid, exhibits levels of steady-state membrane binding equivalent to those of full-length Gag indicates that strong Gag-Gag interaction domains are not required for the efficient binding of HIV-1 Gag to membrane.

Evidence for protein splicing in the endoplasmic reticulum-Golgi intermediate compartment.

We present evidence that the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) is a site for protein splicing of cathepsin C: (i) maturation of the enzyme in COS 7 cells is a two-step process starting within the ERGIC, and (ii) the intermediately processed polypeptide retains both termini of the proenzyme and lacks an internal fragment.

Genomic organization of the mouse reelin gene.

Reelin is the protein defective in reeler mice, an extensively studied model of brain development. The reelin gene (symbol Reln) codes for a protein of the extracellular matrix that contains eight successive repeats of 350 to 390 amino acids. In this work, we describe the genomic structure of the mouse reelin gene and the 5'-flanking genomic DNA sequences. The reelin gene is composed of 65 exons spread over approximately 450 kb of genomic DNA. We identified different reelin transcripts, formed by alternative splicing of a microexon as well as by use of two different polyadenylation sites. All splice sites conform to the GT-AG rule, except for the splice donor site of intron 30, which is GC instead of GT. A processed pseudogene is present in intron 42. Its nucleotide sequence is 86% identical to the sequence of the rat RDJ1 cDNA, which codes for a DnaJ-like protein of the Hsp40 family. Comparison of 8 intron positions in mouse and human reelin genes reveals a highly conserved genomic structure, suggesting a similar structure of the whole gene in both species. We identified two transcription start sites embedded within a CpG. The promoter region contains putative recognition sites for the transcription factors Sp1 and AP2 but lacks TATA and CAAT boxes. The presence of tandemly repeated regions in the Reelin protein suggests that gene duplication events occurred during evolution. By comparison of the amino acid sequences of the eight repeats and the positions of introns, we suggest a model for the evolution of the repeat coding portion of the reelin gene from a putative ancestral minigene.

[Our experience in treating sub crustam skin burns]. / Nashiiat opit ot lechenieto na kozhnite izgarianiia sub crustam.

Alcoholic solution of chlorhexidine and methylene blue was used in 64 patients for local treatment of burned skin surfaces. The wounds healed under the crust, formed from denatured and desiccated fibrin coats and necrotic tissues. The two antiseptic agents (chlorhexidine and methylene blue) provided long-term antibacterial effect. The results were encouraging; epithelialization of rather deep burns was shortened by 4 or 5 days.

[Insertion of the cos-site into DNA of phage M13 and its packing in proteins of phage lambda]. / Vvedenie cos-uchastka v DNK faga M13 i ee upakovka v belki faga lambda.

The cos-site of lambda phage from pHC79 cosmide is transferred to DNA from M13 mp18 phage. The recombinant DNA thus obtained (MC18) is efficiently packaged into lambda proteins in vitro. The BamHI-HindIII fragment of pGP588 (a pBR322 derivatives containing fragment of human DNA) is subcloned into MC18. Although this pGP588 fragment contains numerous Alu repeats, no essential rearrangements of the insert were revealed. The efficiency infection by recombinant DNA packaged with lambda proteins is about 1 X 10(5) pfu/microgram DNA, whereas in the similar conditions the efficiency of lambda EMBL3A was 1 X 10(6) pfu/microgram. It is assumed that the MC vectors might be suitable for cloning and sequencing large fragments either with cohesive or blunt ends. It opens also the way to construct genomic libraries in single-stranded phages.

Protein HMG1 is different from a DNA helix unwinding protein in calf thymus.

A number of criteria were used--chromatography on columns with single-stranded and double-stranded DNA, electrophoresis, peptide analysis, immunological tests and thermal denaturation of DNA--to show that protein (high mobility group) HMG1 and an unwinding protein from calf thymus are two distinct, unrelated proteins. While both proteins are thought to be related to DNA replication this might involve different mechanisms of action.

Isolation of high-mobility-group proteins HMG1 and HMG2 in non denaturing conditions and comparison of their properties with those of acid-extracted proteins.

We describe a method for isolation and purification of the chromosomal proteins HMG1 and HMG2 in non-denaturing conditions which overcomes the difficulties of the published methods concerning yield and purity. The method is based on salt extraction, selective precipitation with ammonium sulfate and DEAE-cellulose chromatography. All studied properties of these proteins (formation of protein tetramers, enhancement of micrococcal nuclease digestion of DNA and chromatin, and protection of 165-basepair DNA in chromatosome) differ significantly from the properties of HMG1 and 2 isolated under denaturing conditions.