The 5′-flanking region of the E1 α form of the murine p16 (MTS1) gene

We have PCR-amplified and sequenced the immediate (841 bp) 5′-flanking region of murine p16^INK4a (MTS1, CDKN2) tumor suppressor gene. Comparing to recently published 5'-flanking region of the human α form of p16^INK4a, homologies were found in several regions of murine p16^INK4a-α putative promoter sequence.     

Cloning and developmental regulation of α1 acid glycoprotein in swine

A cDNA clone of porcine alpha₁ acid glycoprotein (α₁AGP) has been isolated and sequenced. Sequence homologies between porcine, human, and rat indicate that porcine α₁AGP is similar in structure to the rat and human proteins. RNA blots from days 40, 60, 80, and 110 fetal, newborn, and adult livers showed that α₁AGP mRNA is relatively abundant throughout fetal development, particularly at the later stages and in the newborn; there is a rapid decline in abundance following birth. From birth to 3 days of age, there is a three- to four-fold decline in abundance, and α₁AGP mRNA is approximately 100 times less abundant in the adult liver than in that of perinatal pigs. Southern blots showed that α₁AGP is probably a single-copy gene. The isolation of a cloned cDNA for porcine α₁AGP provides a tool to investigate the molecular mechanisms involved in the developmental regulation of the gene and to correlate changes in gene expression during development with fetal growth and well being.     

Assembly of an adult type acetylcholine receptor in a mouse cell line transfected with rat muscle ε-subunit DNA

The mouse muscle cell line BC3H-1 expresses an acetylcholine receptor (AChR) composed of -,β-, and δ-subunits [1]. The functional characteristics of this AChR are comparable to the non-synaptic AChR subtype in mouse muscle [2,3]. To investigate the role of the ε-subunit, which is believed to replace the γ-subunit in forming the adult AChR subtype [4], BC3H-1 cells were stably transfected with cDNA encoding the rat muscle AChR ε-subunit. Expression of this cDNA was under the control of a heat shock promoter, and the plasmid carried the neomycin resistance gene for selection. Several clones were isolated that had integrated the plasmid DNA in a stable form and produced ε-subunit specific RNA after heat induction. Single-channel current recording from cells which contained abundant ε-subunit mRNA identified a novel AChR channel having a larger conductance than the native AChR in these cells. These results suggest that the rat muscle ε-subunit may assemble with mouse muscle -, β- and δ-subunits to form a mouse-rat hybrid AChR with properties similar to that of end-plate channels in the mature mammalian neuromuscular synapse. The novel AChR channel appears in the surface membrane within a few hours following the rise in ε-subunit mRNA. Thus, the notion that replacement of the γ-subunit by the ε-subunit during development is the result of the postnatal rise in the level of ε-subunit specific mRNA is further supported.     

Genetic regulation of skeletal muscle development

During development, skeletal muscles are established in a highly organized manner, which persists throughout life. Molecular and genetic experiments over the last decades have identified many developmental control genes critical for skeletal muscle formation. Developmental studies have shown that skeletal muscles of the body, limb and head have distinct embryonic and cellular origin, and the genetic regulation at work in these domains and during adult myogenesis are starting to be identified. In this review we will summarize the current knowledge on the regulatory circuits that lead to the establishment of skeletal muscle in these different anatomical regions.     

Structure and sequence of the human α- -iduronidase gene

In humans, a deficiency of the lysosomal hydrolase α- -iduronidase (IDUA; EC 3.2.1.76) results in the lysosomal storage of the glycosaminoglycans heparan sulfate and dermatan sulfate, thereby causing the lysosomal storage disorder mucopolysaccharidosis type I. The gene for IDUA is split into 14 exons spanning approximately 19 kb. We report the sequence of two noncontiguous segments of the IDUA gene, one 1.8-kb segment containing exons 1 and 2 and surrounding sequences and a second segment of 4.5 kb containing the last 12 exons. The potential promoter for IDUA has only GC box type consensus sequences consistent with a housekeeping promoter and is bounded by an Alu repeat sequence. The first two exons of IDUA are separated by an intron of 566 bp, then there is a large intron of approximately 13 kb, and the last 12 exons are clustered within 4.5 kb. No consensus polyadenylation signal was found in the 3′ untranslated region, although two variant polyadenylation signals are proposed.     

Expression and regulation of α1β1 integrin in Schwann cells

The interaction of cells with the extracellular matrix plays a critical role in morphogenesis and cell differentiation. To define how Schwann cells might interact with the extracellular matrix, we chose to study the expression of the laminin/collagen receptor α1β1 integrin during nerve development in the rat from embryonic day 14 to maturity. We found that this integrin is expressed predominantly on mature non-myelin-forming cells and only at very low levels on myelin-forming cells. Significant levels of this integrin were not detected on Schwann cell precursors or embryonic Schwann cells in vivo. Experiments using transected and crushed sciatic nerve showed that α1β1 integrin expression is regulated at least in part by axonal contact. Furthermore, Schwann cell culture experiments showed that α1β1 integrin levels are strongly upregulated by transforming growth factor-βs and phorbol esters. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 914–928, 1997     

Replication of single-stranded porcine circovirus DNA by DNA polymerases α and δ

Porcine circovirus is the only mammalian DNA virus so far known to contain a single-stranded circular genome (Tischer et al. (1982) Nature 295, 64–66). Replication of its small viral DNA (1.76 kb) appears to be dependent on cellular enzymes expressed during S-phase of the cell cycle (Tischer et al. (1987) Arch. Virol. 96, 39–57). In this paper we have exploited the porcine circovirus genome to probe for in vitro initiation and elongation of DNA replication by different preparations of calf thymus DNA polymerase α and δ as well as by a partially purified preparation from pig thymus. The results indicated that three different purification fractions of calf thymus DNA polymerase α and one from pig thymus initiate DNA synthesis at several sites on the porcine circovirus DNA. It appears that the sites at which DNA primase synthesizes primers are not entirely random. Subsequent DNA elongation by a highly purified DNA polymerase α holoenzyme which had been isolated by the criterion of replicating single-stranded M13 DNA (Ottiger et al. (1987) Nucleic Acids Res. 15, 4789–4807) is very efficient. Complete conversion to the double-stranded form is obtained in less than 1 min. When the DNA synthesis by DNA polymerase α is blocked with the DNA polymerase α specific monoclonal antibody SJK 132-20 after initiation by DNA primase, DNA polymerase δ can efficiently replicate from the primers. This in vitro DNA replication system may be used in analogy to the bacteriophage systems in E. coli to study initiation and elongation of DNA replication.     

Dissecting α-helices: Position-specific analysis of α-helices in globular proteins

An analysis of the amino acid distributions at 15 positions, viz., N“, N′, Ncap, N1, N2, N3, N4, Mid, C4, C3, C2, C1, Ccap, C′, and C” in 1,131 α-helices reveals that each position has its own unique characteristics. In general, natural helix sequences optimize by identifying the residues to be avoided at a given position and minimizing the occurrence of these avoided residues rather than by maximizing the preferred residues at various positions. Ncap is most selective in its choice of residues, with six amino acids (S, D, T, N, G, and P) being preferred at this position and another 11 (V, I, F, A, K, L, Y, R, E, M, and Q) being strongly avoided. Ser, Asp, and Thr are all more preferred at Ncap position than Asn, whose role at helix N-terminus has been highlighted by earlier analyses. Furthermore, Asn is also found to be almost equally preferred at helix C-terminus and a novel structural motif is identified, involving a hydrogen bond formed by N^δ2 of Asn at Ccap or C1 position, with the backbone carbonyl oxygen four residues inside the helix. His also forms a similar motif at the C-terminus. Pro is the most avoided residue in the main body (N4 to C4 positions) and at C-ter-minus, including Ccap of an α-helix. In 1,131 α-helices, no helix contains Pro at C3 or C2 positions. However, Pro is highly favoured at N1 and C′. The doublet X-Pro, with Pro at C′ position and extended backbone conformation for the X residue at Ccap, appears to be a common structural motif for termination of α-helices, in addition to the Schellman motif. Main body of the helix shows a high preference for aliphatic residues Ala, Leu, Val, and Ile, while these are avoided at helix termini. A propensity scale for amino acids to occur in the middle of helices has been obtained. Comparison of this scale with several previously reported scales shows that this scale correlates best with the experimentally determined values. Proteins 31:460–476, 1998. © 1998 Wiley-Liss, Inc.     

Chromosomal localization of the human interleukin 1α (IL-1α) gene

The human interleukin 1α gene was assigned to chromosome 2 using Southern transfer analysis of human-rodent somatic cell hybrid DNAs. The gene was regionally localized to 2q12–21 using in situ hybridization to metaphase chromosomes. These results indicate that the IL-1α gene maps to the same general region on the long arm of chromosome 2 as the IL-1β gene, which has been previously assigned.     

Isolation and sequencing of porcine fast skeletal muscle alkali myosin light chain 3 cDNA

Abstract

A cDNA coding for alkali myosin light chain 3 (MLC3_F) was isolated from a porcine skeletal muscle library. This clone has an insert of 859 bp encompassing the complete CDS (coding sequence) plus the 5’ and 3’ untranslated regions. Computer analysis showed that porcine MLC3_F cDNA is highly homologous to the corresponding cDNAs of human, rabbit and rat. Moreover, Northern analysis showed the presence of two bands that represent the mature mRNAs of the MLCl_F and MLC3_F isoforms according to data observed in other species.     

Structure and evolution of human α-fetoprotein deduced from partial sequence of cloned cDNA

The nucleotide sequence of a recombinant DNA clone, containing a partial mRNA sequence for human α-fetoprotein (AFP) in the plasmid vector pBR322, has been determined. Two regions of the cloned nucleotide sequence were found to agree with published amino acid sequences of two cyanogen bromide peptides derived from human AFP. Examination of the amino acid sequence, deduced from the cloned portion of the mRNA coding region, reveals extensive homology with the third domain of the human serum albumin molecule. A total of 44% ( ) amino acids and 54% ( ) nucleotides are identical in the two structures. The landmark cysteine residues are found in the same positions in both polypeptide chains, presumably forming the same disulfide bridges in AFP as those found in the albumin. The sequence homology reinforces the evidence that human AFP and albumin constitute a gene family, in analogy to the same family found in rodents. A comparison of the human and rodent sequence data suggests that the rate of molecular evolution has been faster for AFP than for albumin.