The dacA gene of Bacillus stearothermophilus coding for D-alanine carboxypeptidase: cloning, structure and expression in Escherichia coli and Pichia pastoris.

The bacterial D-alanine carboxypeptidases (CPases) remove C-terminal D-alanyl residues from sugar-peptide cell wall precursors. The CPases have many characteristics in common with the high-M(r) penicillin-binding proteins (PBPs) whose inhibition by beta-lactam antibiotics is lethal. The CPases are attractive as model PBPs, because of their relatively lower M(r) and higher activity in vitro. We have cloned and sequenced the Bacillus stearothermophilus gene (dacA) coding for a membrane-bound CPase. The nucleotide (nt) sequence of the gene is homologous to that of the Escherichia coli and Bacillus subtilis dacA loci, which also code for membrane-bound CPases. E. coli host cells lysed when expression of B. stearothermophilus dacA was induced. The same coding sequence was expressed in the methylotrophic yeast, Pichia pastoris, using the alcohol oxidase-1 (AOX1) promoter. Over 100 micrograms/ml of CPase was efficiently secreted into the medium after induction by methanol, without adversely affecting this host. The yeast product is indistinguishable from the native enzyme in structure and activity. The ability to secrete large amounts of heterologous protein and the lack of endogenous peptidoglycan metabolism makes P. pastoris an attractive candidate for the production of PBPs.

Polyclonal B cell hyperplasia associated with Epstein-Barr virus causing acute renal allograft failure.

Six weeks post cadaver renal transplantation, a patient developed a flu-like illness. Acute renal failure unresponsive to anti-rejection therapy occurred and he died four days later from Pneumocystis carinii pneumonia and Streptococcus viridans septicemia. Autopsy revealed a diffuse polymorphic polyclonal B cell infiltrate occupying most organs, including the allograft. Primary Epstein-Barr Virus (EBV) infection was established by 1) rising anti-EBV antibody titres; 2) the demonstration of EBV nuclear antigen in the infiltrate and 3) the presence of EBV specific DNA sequences in affected tissues. EBV associated polymorphic B cell hyperplasia can mimic rejection and result in acute allograft failure.

Internal structure of the silk fibroin gene of Bombyx mori. II. Remarkable polymorphism of the organization of crystalline and amorphous coding sequences.

Alleles of the silk fibroin locus from 22 inbred stocks of Bombyx mori were compared. Nineteen alleles differing from one another in length and internal sequence organization were distinguished. Individuals from a single stock generaly are homozygous for a particular allele, as judged by their gene restriction pattern and the length of the fibroin protein produced. Restriction with endonucleases having four base recognition sequences revealed no variation with respect to these particular coding sequences among the alleles tested. Furthermore, digestion with endonucleases specific for amorphous coding sequences indicated that all the alleles tested had amorphous coding sequence domains alternating regularly with crystalline domains just as was found for the L allele. The stocks differed considerably in their fibroin length, and in the total length of the fibroin coding regions of their genes. These differences were accounted for by variation in the lengths of crystalline coding domains when compared to the ends of the genes. Several characteristics of the alleles indicates that this variation results from recombination between the highly repetitive coding sequences of misaligned genes (homologous unequal crossing-over). Polymorphism of the fibroin gene in B, mori appears to be greater than for any other gene for which data are available.

Internal structure of the silk fibroin gene of Bombyx mori. I The fibroin gene consists of a homogeneous alternating array of repetitious crystalline and amorphous coding sequences.

The DNA sequence orgainzation of the protein encoding region of the gene for silk fibroin has been analyzed. The accompanying paper (Manningm R. F., and Gage, L. P. (1980) J. Biol. Chem. 255, 9451-9457) shows that the total length of the gene, and its protein, as well as the pattern of restriction sites in the gene is highly polymorphic among inbred stocks of Bombyx mori, In this paper, those features of fibroin gene structure which are invariant among these alleles are presented. Fibroin is composed primarily of relatively short "crystalline" and "amorphous" peptides of known sequence whose arrangement in the protein is unknown. Knowledge of the codons most commonly used in fibroin mRNA allowed utilization of particular restriction inzymes as a means for determing the nature and organization of crystalline and amorphous coding sequences in the fibroin gene. Three restriction endonucleases were identified that cleve sequences coding for amorphous region peptides. Their cleavage pattern revelaed that the repetitive coding sequence of the gene core (approximately 15 kilobases) is divided into at least 10 large crystalline coding domains interrupted by smaller amorphous coding domains. Many restriction endoncleases do not cleave the fibroin core at all, three of them with four gase recognition sequences. Specific deductions as to codon usage and repetitive sequence homogeneity in the gene follow from these results. One novel finding is the rigorous exclusion of the glycine codon GGA prior to serine codons even though this glycine codon is used frequently prior to alanine codons. The sequence homogeneity and the regularly alternating arrangement of crystalline and amorphous coding sequences of the gene are discussed in terms of the function of fibroin protein and the evolution of highly repetitive DNA.

Alleles of the fibroin gene coding for proteins of different lengths.

Bombyx mori silkworms producing fibroin proteins of different lengths have been analyzed genetically and shown to possess variant alleles of a single fibroin gene. The structures of two alleles have been compared by using restriction endonuclease sites inside and outsite the fibroin gene as physical markers. We find that fibroins distinguishable on the basis of length are encoded by genes with different internal structures and overall lengths. Our results strongly support the idea that rearrangements within the highly repetitive sequences of the fibroin gene are the result of unequal recombination, and can give rise to variant fibroin genes with altered coding lengths.

The genes for 18S, 5.8S and 28S ribosomal RNA of Bombyx mori are organized into tandem repeats of uniform length.

The organization of the multiple genes for 18S, 5.8S and 28S rRNA in the genome of the silkworm, Bombyx mori was determined by restriction endonuclease digestion and Southern blot hybridization. The ribosomal genes (rDNA) are tandemly reiterated, with a uniform repeat length of 6.9 . 10(6) daltons. Each rDNA repeat has a single site for EcoRI, HindIII, HpaI and SmaI and each of these sites has been mapped with respect to the others and to the rRNA genes; each repeat consists of a transcribed region (6 . 10(6)daltons) containing the 18S, 5.8S and 28S rRNA genes (5' leads to 3') and also a small non-transcribed spacer (approximately 10(6) daltons). Complete rDNA repeats were cloned using the vector RSF2124 and grown in Escherichia coli. Characterization of the rDNA plasmids confirmed the conclusions from studies of the total rDNA. The organization of B. mori rDNA is similar to that of other eukaryotes, except for the absence of heterogeneity in the rDNA repeat length; thus, there is neither variation in the length of the non-transcribed spacer nor the presence of inserts in a detectable portion of the rDNA. The utility of this map, and particularly of the rDNA plasmids, for detailed studies of rRNA transcription and processing is discussed.

Physical map of the Bombyx mori DNA containing the gene for silk fibroin.

A physical map of the DNA containing the gene for silk fibroin was developed from direct hybridization analysis of restriction endonuclease digests of total Bombyx mori DNA using fibroin 125I-mRNA. The orientation of mRNA transcription relative to this map was deduced from the sensitivity of the mRNA coding strand within certain DNA restriction segments to lambda-exonuclease and exonuclease III. The map includes the entire gene coding region (Mr approximately 11 x 10(6)) and large DNA elements which flank the gene at its 5' end (Mr approximately 3 x 10(6)) and 3' end (Mr approximately 6.5 x 10(6)). The coding region is remarkably uniform in its sensitivity to restriction endonucleases. It is completely devoid of sites for most of the enzymes tested, including Hae III, the recognition sequence (d-pG-G C-C) of which might be expected to occur frequently in this large DNA block of 60% G + C content. The fibroin coding region does contain an enormous number of sites for enzymes predicted to have activity from known fibroin mRNA sequences. These results suggeste that the fibroin gene core is a large homogeneously repetitive block of DNA with little evidence for sequence divergence, or the presence of qualitatively different sequences, which might creat other restriction sensitivities. The map also allowed a comparison to be made of the fibroin gene "context" in DNA from tissues either active or inactive in fibroin synthesis.

The blocked and methylated 5' terminal sequence of a specific cellular messenger: the mRNA for silk fibroin of Bombyx mori.

The messenger RNA for silf fibroin, laveled with 32PO4 and methyl-3H L-methionine, was purified to near homogeneity from the posterior silk gland of the sildworm Bombyx mori, and the sequence of a methylated, RNAase T2-resistant structure was determined. This sequence is similar structurally to 5' terminal blocked and methylated sequences found on the total populations of polyadenylated eucaryotic cellular and certain viral mRNAs. The RNAase T2-resistant oligomer from fibroin mRNA was cleaved by nucleases P1 into three components: a blocked and methylated sequence containing three phosphates; a 2'-0-methyl UMP residue (pUm), and an unmethylated CMP (pC). The blocked and methylated sequence comigrated in three chromatographic systems with the blocked and methylated terminus of silkworm cytoplasmic polyhedrosis virus mRNA, which has the structure m7GpppAm. The fibroin mRNA cap was cleaved by nucleotide pyrophosphatase to yeild 7-methyl GMP Tpm7G) and 2'-0-methyl AMP (pAm). This sequence also appeared to be terminally located, with the m7G joined by a 5'-5' pyrophosphate linkage to the Am. It was concluded that the 5' terminal sequence of fibroin mRNA molecules is m7G(5')ppp(5')AmpUmpCp. The regulation of expression of the highly specialized gene for fibroin is discussed in light of this finding.