Vitellogenin and yolk protein processing in Bothrops jararaca (Wied), a tropical venomous snake.

The plasma vitellogenin of Bothrops jararaca is composed of two subunits. The larger subunit (160 kDa) is phosphate rich and carbohydrate poor, while the smaller (110 kDa) is highly glycosylated and less phosphorylated. As in other vertebrates, the vitellogenin of B. jararaca is synthesized in the liver under estrogen control. The newly synthesized vitellogenin molecule is a 270 kDa polypeptide. This polypeptide originates the two subunits of the plasma vitellogenin by proteolytic cleavage. In the eggs of B. jararaca six main yolk polypeptides have been detected (113, 107, 104, 72, 27.2 and 20.7 kDa). Using phosphoprotein staining we have shown that the 72 kDa polypeptide is the larger phosvitin so far described in a vertebrate egg yolk. The 107 kDa yolk polypeptide also seems to be phosphorylated, but to a lesser extent than the phosvitin. The 104 kDa vitellin originates from the larger vitellogenin subunit while the 113 kDa vitellin originates from the smaller vitellogenin subunit. Based on these results we propose a general scheme for vitellogenin and vitellin processing in B. jararaca.

Molecular characterization of the C-3 DNA puff gene of Rhynchosciara americana.

We have mapped a region of about 33 kb which includes the transcription unit of the C-3 DNA puff gene of Rhynchosciara americana. The C-3 TU and a region extending approximately 800 bp upstream of the C-3 promoter were characterized. The TU is composed of three exons and produces a 1.1-kb mRNA whose level in salivary glands increases with the expansion of the C-3 puff. The C-3 messenger appears to undergo rapid deadenylation resulting in an RNA of about 0.95 kb which can still be observed in gland cells 15 h after the puff has regressed. The 1.1-kb mRNA codes for a 32.4-kDa, predominantly alpha-helical polypeptide with three conserved parallel coiled-coil stretches. The aa composition and structure of this polypeptide suggests that it is secreted and contributes to the formation of the cocoon in which the larvae pupate. The region upstream of the promoter contains several A-rich sequences with similarity to the ACS of yeast which might have a role in the initiation of replication/amplification.

Comparison of a vitellogenin gene between two distantly related rhabditid nematode species.

Three vitellogenin genes from the free-living nematode Caenorhabditis elegans have previously been characterized at the molecular level. In order to study evolutionary relationships within this poorly understood taxon, we have cloned a vitellogenin gene, CEW1-vit-6, from a distantly related species belonging to the same family as C. elegans. Screening of a genomic library with a probe to total poly(A+) RNA yielded three clones that hybridized more intensely than all others, and all three corresponded to a single gene homologous to C. elegans vit-6. Comparison of CEW1-vit-6 with Ce-vit-6 reveals both strong similarities and surprising differences. Life Ce-vit-6, the gene is about 5 kb long and contains four unusually small introns (38-41 nt), but only one interrupts the gene at the same location as a Ce-vit-6 intron. The promoter region contains five matches to Vitellogenin Promoter Element 1 (VPE1) and no matches to VPE2, both previously shown to be required for vit gene transcription in C. elegans. Codon usage is in general similar to that of the Ce-vit genes, but a few codon biases are quite different. Alignment of the CEW1-vit-6 protein with Ce-vit-6 and Ce-vit-2 products suggests the existence of two domains which have evolved at different rates. Sequence comparison shows that nematode vitellogenins are much more closely related to vertebrate than to insect vitellogenins.

The effect of pH and metal chelators upon the sedimentation velocity of Odontophrynus americanus vitellogenin

In the tetraploid amphibian Odontophrynus americans the selective precipitation of vitellogenin by Mg2+ from plasma treated with ethylene diaminetetraacetic acid (EDTA) or ethylene bis (oxyethylenenitrilo)-tetraacetic acid (EGTA) is a pH-dependent phenomenon. Utilizing sucrose gradient centrifugation of whole plasma we have shown that under standardconditions (pH 7.0) the estimated apparent sedimentation coefficient of vitellogenin is 17s.At pH 8.0 and in the presence of EDTA or EGTA there is a decrease of the vitellogenin sedimentation coefficient.This behavior is restricted to vitellogenin as othewr plasma proteins show no alteration in their sedimentation coefficient after similar treatment. The treatment with EDTA at pH 8.0 also induces changes in the vitellogenin molecule which can be detected by partial proteolysis with chymotripsin A

The effect of pH and metal chelators upon the sedimentation velocity of Odontophrynus americanus vitellogenin.

1. In the tetraploid amphibian Odontophrynus americanus the selective precipitation of vitellogenin by Mg2+ from plasma treated with ethylene diaminetetraacetic acid (EDTA) or ethylene bis (oxyethylenenitrilo)-tetraacetic acid (EGTA) is a pH-dependent phenomenon. 2. Utilizing sucrose gradient centrifugation of whole plasma we have shown that under standard conditions (pH 7.0) the estimated apparent sedimentation coefficient of vitellogenin is 17S. At pH 8.0 and in the presence of EDTA or EGTA there is a decrease of the vitellogenin sedimentation coefficient. This behavior is restricted to vitellogenin as other plasma proteins show no alteration in their sedimentation coefficient after similar treatment. 3. The treatment with EDTA at pH 8.0 also induces changes in the vitellogenin molecule which can be detected by partial proteolysis with chymotrypsin A.

In vivo effects of ecdysterone on puff formation, and RNA and protein synthesis in the salivary glands of Rhynchosciara americana.

1. Fourth-instar larvae of Rhynchosciara americana were injected with the insect molting hormone, ecdysterone, giving final hemolymph concentrations from 4.46 to 223 microM. 2. Induction of the DNA puff, B2b, in the proximal (S1) region of the salivary glands of Rhynchosciara americana by 22.6 microM ecdysterone, was accompanied by the production of an mRNA and a polypeptide with the same characteristics as B2b products produced during normal development. This mRNA and polypeptide were restricted to the proximal region of the gland, as is the B2b puff. 3. Synthesis of other poly(A)+RNAs was also stimulated in S1 by ecdysterone, and other puffs that appear during normal development were induced. However, rRNA production in S1 goes through a pattern of inhibition, followed by recovery when B2b is puffed, and subsequent inhibition. 4. Low molecular weight RNA, with a peak in the region of 4S, is stimulated after ecdysterone administration.

In vivo effects of ecdysterone on puff formation, and RNA and protein synthesis in the salivary glands of Rhynchosciara americana

1. Fourth-instar larvae of Rhynchosciara americana were injected with the insect molting hormone, ecdysterone, giving final hemolymph concentrations from 4.46 to 223µM. 2. Induction of the DNA puff, B2b, in the proximal (S1) region of the salivary glands of Rhynchosciara americana by 22.6 µM ecdyesterone, was accompanied by the production of an mRNA and a polypeptide with the same characteristics as B2b products produced during normal development. This mRNA and polypeptide were restricted to the proximal region of the gland, as is the B2b puff. 3. Synthesis of other poly (A) +RNAs was also stimulated in S1 by ecdysterone, and other puffs that appear during normal development were induced. However, rRNA production in S1 goes through a pattern of inhibition, followed by recovery when B2b is puffed, and subsequent inhibition. 4. Low molecular weight RNA, with a peak in the region of 4S, is stimulated after ecdysterone administration